Nucleic acid amplification methods

ABSTRACT

The present invention relates to assays and kits for carrying out said assays for the rapid, automated detection of infectious pathogenic agents and normal and abnormal genes. The present invention further relates to methods for general amplification of total mRNAs and for analyzing differential mRNA expression using the amplification methods disclosed herein.

The present application is a Divisional of U.S. application Ser. No.09/728,265 filed Dec. 1, 2000, which is a continuation-in-part of U.S.application Ser. No. 09/299, 217, filed Apr. 23, 1999, now U.S. Pat. No.6,569,647 which is a continuation of U.S. application Ser. No.08/690,494 filed Jul. 31, 1996, now U.S. Pat. No. 5,942,391 which is acontinuation-in-part of PCT/US95/07671, filed Jun. 4, 1995 which is acontinuation-in-part of U.S. application Ser. No. 08/263,937, filed Jun.22, 1994, now abandoned.

INTRODUCTION

The present invention relates to assays and kits for carrying out saidassays for the rapid, automated detection of infectious pathogenicagents and normal and abnormal genes. The present invention furtherrelates to methods for general amplification of genomic DNA and totalmRNAs and for analyzing differential mRNA expression using theamplification methods disclosed herein.

BACKGROUND OF THE INVENTION

A number of techniques have been developed recently to meet the demandsfor rapid and accurate detection of infectious agents, such as viruses,bacteria and fungi, and detection of normal and abnormal genes. Suchtechniques, which generally involve the amplification and detection (andsubsequent measurement) of minute amounts of target nucleic acids(either DNA or RNA) in a test sample, include inter alia the polymerasechain reaction (PCR) (Saiki, et al., Science 230:1350, 1985; Saiki etal., Science 239:487, 1988; PCR Technology, Henry A. Erlich, ed.,Stockton Press, 1989; Patterson et al., Science 260:976, 1993), ligasechain reaction (LCR) (Barany, Proc. Natl. Acad. Sci. USA 88:189, 1991),strand displacement amplification (SDA) (Walker et al., Nucl. Acids Res.20:1691, 1992), Qβ replicase amplification (QβRA) (Wu et al., Proc.Natl. Acad. Sci. USA 89:11769, 1992; Lomeli et al., Clin. Chem. 35:1826,1989) and self-sustained replication (3SR) (Guatelli et al., Proc. Natl.Acad. Sci. USA 87:1874-1878, 1990). While all of these techniques arepowerful tools for the detection and identification of minute amounts ofa target nucleic acid in a sample, they all suffer from variousproblems, which have prevented their general applicability in theclinical laboratory setting for use in routine diagnostic techniques.

One of the most difficult problems is preparation of the target nucleicacid prior to carrying out its amplification and detection. This processis time and labor intensive and, thus, generally unsuitable for aclinical setting, where rapid and accurate results are required. Anotherproblem, especially for PCR and SDA, is that conditions for amplifyingthe target nucleic acid for subsequent detection and optionalquantitation vary with each test, i.e., there are no constant conditionsfavoring test standardization. This latter problem is especiallycritical for the quantitation of a target nucleic acid by competitivePCR and for the simultaneous detection of multiple target nucleic acids.

Circumvention of the aforementioned problems would allow for developmentof rapid standardized assays, utilizing the various techniques mentionedabove, that would be particularly useful in performing epidemiologicinvestigations, as well as in the clinical laboratory setting fordetecting pathogenic microorganisms and viruses in a patient sample.Such microorganisms cause infectious diseases that represent a majorthreat to human health. The development of standardized and automatedanalytical techniques and kits therefor, based on rapid and sensitiveidentification of target nucleic acids specific for an infectiousdisease agent would provide advantages over techniques involvingimmunologic or culture detection of bacteria and viruses.

Reagents may be designed to be specific for a particular organism or fora range of related organisms. These reagents could be utilized todirectly assay microbial genes conferring resistance to variousantibiotics and virulence factors resulting in disease. Development ofrapid standardized analytical techniques will aid in the selection ofthe proper treatment.

In some cases, assays having a moderate degree of sensitivity (but highspecificity) may suffice, e.g., in initial screening tests. In othercases, great sensitivity (as well as specificity) is required, e.g., thedetection of the HIV genome in infected blood may require finding thevirus nucleic acid sequences present in a sample of one part per 10 to100,000 human genome equivalents (Harper et al., Proc. Nat'l. Acad.Sci., USA 83:772, 1986).

Blood contaminants, including inter alia, HIV, HTLV-I, hepatitis B andhepatitis C, represent a serious threat to transfusion patients and thedevelopment of routine diagnostic tests involving the nucleic acids ofthese agents for the rapid and sensitive detection of such agents wouldbe of great benefit in the clinical diagnostic agree laboratory. Forexample, the HIV genome can be detected in a blood sample using PCRtechniques, either as an RNA molecule representing the free viralparticle or as a DNA molecule representing the integrated provirus (Ouet al, Science 239:295, 1988; Murakawa et al., DNA 7:287, 1988).

In addition, epidemiologic investigations using classical culturingtechniques have indicated that disseminated Mycobacteriumavium-intracellulaire (MAI) infection is a complication of late-stageAcquired Immunodeficiency Syndrome (AIDS) in children and adults. Theprecise extent of the problem is not clear, however, since currentcultural methods for detecting mycobacteria are cumbersome, slow and ofquestionable sensitivity. Thus, it would be desirable and highlybeneficial to devise a rapid, sensitive and specific technique for MAIdetection in order to provide a definitive picture of the involvement inHIV-infected and other immunosuppressed individuals. Such studies mustinvolve molecular biological methodologies, based on detection of atarget nucleic acid, which have routinely been shown to be moresensitive than standard culture systems (Boddinghaus et al., J. Clin.Med. 28:1751, 1990).

Other applications for such techniques include detection andcharacterization of single gene genetic disorders in individuals and inpopulations (see, e.g., Landergren et al., Science 241: 1077, 1988 whichdiscloses a ligation technique for detecting single gene defects,including point mutations). Such techniques should be capable of clearlydistinguishing single nucleotide differences (point mutations) that canresult in disease (e.g., sickle cell anemia) as well as deleted orduplicated genetic sequences (e.g., thalassemia).

The methods referred to above are relatively complex procedures that, asnoted, suffer from drawbacks making them difficult to use in theclinical diagnostic laboratory for routine diagnosis and epidemiologicalstudies of infectious diseases and genetic abnormalities. All of themethods described involve amplification of the target nucleic acid to bedetected. The extensive time and labor required for target nucleic acidpreparation, as well as variability in amplification templates (e.g.,the specific target nucleic acid whose detection is being measured) andconditions, render such procedures unsuitable for standardization andautomation required in a clinical laboratory setting.

The present invention is directed to the development of rapid, sensitiveassays useful for the detection and monitoring of pathogenic organisms,as well as the detection of abnormal genes in an individual. Moreover,the methodology of the present invention can be readily standardized andautomated for use in the clinical laboratory setting.

SUMMARY OF THE INVENTION

An improved method, which allows for rapid, sensitive and standardizeddetection and quantitation of nucleic acids from pathogenicmicroorganisms from samples from patients with infectious diseases hasnow been developed. The improved methodology also allows for rapid andsensitive detection and quantitation of genetic variations in nucleicacids in samples from patients with genetic diseases or neoplasia.

This method provides several advantages over prior art methods. Themethod simplifies the target nucleic acid isolation procedure, which canbe performed in microtubes, microchips or micro-well plates, if desired.The method allows for isolation, amplification and detection of nucleicacid sequences corresponding to the target nucleic acid of interest tobe carried out in the same sample receptacle, e.g., tube or micro-wellplate.

In another aspect of the invention, the techniques described herein maybe used for detection of specific genes or markers at the single celllevel using a gel matrix or slide format. In situ amplification anddetection of nucleic acid sequences in single cells may be carried outusing cells embedded in a semi-solid gel matrix. Such methods can beused to detect a mutation in a single cell, such as a tumor cell, or todetect chromosomal abnormalities in single cells such as embryo cells.

The method also allows for standardization of conditions, because only apair of generic amplification probes may be utilized in the presentmethod for detecting a variety of target nucleic acids, thus allowingefficient multiplex amplification. The method also allows the directdetection of RNA by probe amplification without the need for DNAtemplate production. The amplification probes, which in the method maybe covalently joined end to end, form a contiguous ligated amplificationsequence. The assembly of the amplifiable DNA by ligation increasesspecificity, and makes possible the detection of a single mutation in atarget. This ligated amplification sequence, rather than the targetnucleic acid, is either directly detected or amplified, allowing forsubstantially the same amplification conditions to be used for a varietyof different infectious agents and, thus, leading to more controlled andconsistent results being obtained. In addition, multiple infectiousagents in a single sample may be detected using the multiplexamplification methodology disclosed.

Additional advantages of the present invention include the ability toautomate the protocol of the method disclosed, which is important inperforming routine assays, especially in the clinical laboratory and theability of the method to utilize various nucleic acid amplificationsystems, e.g., polymerase chain reaction (PCR), strand displacementamplification (SDA), ligase chain reaction (LCR) and self-sustainedsequence replication (3SR).

The present method incorporates magnetic separation techniques usingparamagnetic particles or beads coated with a ligand binding moiety thatrecognizes and binds to a ligand on an oligonucleotide capture probe toisolate a target nucleic acid (DNA or RNA) from a sample of a clinicalspecimen containing e.g., a suspected pathogenic microorganism or geneabnormality, in order to facilitate detection of the underlyingdisease-causing agent.

In one aspect of the present invention, a target nucleic acid ishybridized to a pair of non-overlapping oligonucleotide amplificationprobes in the presence of paramagnetic beads coated with a ligandbinding moiety, e.g., streptavidin, to form a complex. These probes arereferred to as a capture/amplification probe and an amplification probe,respectively. The capture/amplification probe contains a ligand, e.g.biotin, that is recognized by and binds to the ligand binding moiety onthe paramagnetic beads. The probes are designed so that each containsgeneric sequences (i.e., not target nucleic acid specific) and specificsequences complementary to a nucleotide sequence in the target nucleicacid. The specific sequences of the probes are complementary to adjacentregions of the target nucleic acid, and thus do not overlap one another.Subsequently, the two probes are joined together using a ligating agentto form a contiguous ligated amplification sequence. The ligating agentmay be an enzyme, e.g., DNA ligase or a chemical. Following washing andremoval of unbound reactants and other materials in the sample, thedetection of the target nucleic acid in the original sample isdetermined by detection of the ligated amplification sequence. Theligated amplification sequence may be directly detected if a sufficientamount (e.g., 10⁶-10⁷ molecules) of target nucleic acid was present inthe original sample. If an insufficient amount of target nucleic acid(<10⁶ molecule) was present in the sample, the ligated amplificationsequence (not the target nucleic acid) may be amplified using suitableamplification techniques, e.g. PCR, for detection. Alternatively,capture and amplification functions may be performed by separate andindependent probes. For example, two amplification probes may be ligatedto form a contiguous sequence to be amplified. Unligated probes, as wellas the target nucleic acid, are not amplified in this technique. Yetanother alternative is a single amplification probe that hybridizes tothe target such that its 3′ and 5′ ends are juxtaposed. The ends arethen ligated by DNA ligase to form a covalently linked circular probethat can be identified by amplification.

The present invention further provides methods for general amplificationof total genomic DNA or mRNA expressed within a cell. The use of suchmethods provides a means for generating increased quantities of DNAand/or mRNA from small numbers of cells. Such amplified DNA and/or mRNAmay then be used in techniques developed for detection of infectiousagents, and detection of normal and abnormal genes.

In addition, the invention provides a novel differential displayligation dependent RAM method for identifying differentially expressedmRNAs within different types of cells.

Further, the invention provides methods wherein thecapture/amplification probe can be designed to bind to an antibody. Forexample, one antibody can be attached to a capture/amplification probeand the other antibody can be attached to a target sequence. In thisinstance only if both antibodies are bound to the same antigen willligation occur. This technique can be used for ELISA in a liquid phaseRAM reaction or in situ in a solid phase RAM reaction.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a generic schematic diagram showing the various componentsused in the present method of capture, ligation-dependent amplificationand detection of a target nucleic acid.

FIG. 2 is a schematic flow diagram generally showing the various stepsin the present method.

FIG. 3 is an autoradiograph depicting the detection of a PCR amplifiedprobe that detects HIV-1 RNA. Lane A is the ligated amplificationsequence according to the invention; Lane B, which is a control, is PCRamplified nanovariant DNA, that does not contain any HIV-1-specificsequences.

FIG. 4 is a schematic diagram of an embodiment of the present inventionshowing the various components used for capture and ligation-dependentdetection of a target nucleic acid, e.g., HCV RNA, and subsequentamplification of its sequences, employing two capture/amplificationprobes containing a bound biotin moiety and two ligation-dependentamplification probes.

FIG. 5 is a schematic flow diagram showing magnetic isolation, targetspecific ligation and PCR amplification for the detection of HCV RNAusing a single capture/amplification probe and two amplification probes.

FIG. 6 is a schematic diagram showing the various components used toamplify and detect a target nucleic acid, e.g., HCV RNA, employing twocapture/amplification probes, each containing a bound biotin moiety, anda single amplification probe.

FIG. 7 is a schematic diagram showing various components used to detecta target nucleic acid, e.g., HCV RNA, employing twocapture/amplification probes, each containing a bound biotin moiety, anda single amplification probe that circularizes upon hybridization to thetarget nucleic acid and ligation of free termini.

FIG. 8 is a photograph of ethidium bromide stained DNA depicting PCRamplified probes used to detect HCV RNA in a sample. The amount of HCVRNA in the sample is determined by comparing sample band densities tothose of standard serial dilutions of HCV transcripts.

FIG. 9 is a photograph of ethidium bromide stained DNA depicting PCRamplified single, full length ligation-dependent and circularizableprobes used to detect HCV RNA in a sample. The amount of HCV RNA in thesample is determined by comparing sample band densities to those ofstandard serial dilutions of HCV transcripts.

FIG. 10 is a schematic diagram illustrating the capture and detection ofa target nucleic acid by the hybridization signal amplification method(HSAM).

FIG. 11 is a schematic diagram illustrating the use of HSAM to detect anantigen with a biotinylated antibody and biotinylated signal probes.

FIGS. 12A and B are schematic diagrams illustrating RNA-proteincrosslinks formed during formalin fixation. FIG. 12A depicts theprevention of primer extension due to the crosslinks in the method ofreverse transcription PCR (RT-PCR). FIG. 13B illustrates thathybridization and ligation of the probes of the present invention arenot prevented by protein-RNA crosslinks.

FIG. 13 is a schematic diagram of multiplex PCR. Two set ofcapture/amplification probes, having specificity for HIV-1 and HCV,respectively, are used for target capture, but only one pair of genericPCR primers is used to amplify the ligated probes. The presence of eachtarget can be determined by the size of the amplified product or byenzyme-linked immunosorbent assay.

FIG. 14 is a schematic diagram of HSAM using a circular target probe andthree circular signal probes. AB, CD and EF indicate nucleotidesequences in the linker regions that are complementary to the 3′ and 5′nucleotide sequences of a circular signal probe. AB′, CD′ and EF′indicate the 3′ and 5′ nucleotide sequences of the signal probes thathave been juxtaposed by binding to the complementary sequences of thelinker regions of another circular signal probe.

FIG. 15 is a schematic diagram of HSAM utilizing a circular target probeand linear signal probes.

FIG. 16 is a schematic diagram of amplification of a circularized probeby primer-extension/displacement and PCR.

FIG. 17 is a schematic diagram of an embodiment of RAM in which a T3promoter has been incorporated into Ext-primer 2, allowing amplificationof the circular probe by transcription.

FIG. 18 provides a polyacrylamide gel depicting the amplification of acircular probe by extension of Ext-primer 1.

FIG. 19 is a schematic diagram of amplification of a circularized probeby the ramification-extension amplification method (RAM).

FIG. 20 is a diagram of a RAM assay in which an RNA polymerase promotersequence is incorporated into the primer.

FIG. 21 depicts a RAM assay in the presence of 1, 2 and 3 primers.

FIG. 22 is a schematic diagram of a RAM assay with serial dilution oftarget DNA.

FIG. 23 depicts a RAM assay where target sequences of increased lengthsare amplified.

FIG. 24 depicts the capture of a target nucleic acid on a solid supportutilizing a circular probe.

FIG. 25 is a diagram of the detection of an antibody or antigen using acapture/primer that specifically binds to the antibody or antigen.

FIG. 26 depicts the genetic amplification of genomic DNA using adaptormolecules.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed towards simplified sample preparationand generic amplification systems for use in clinical assays to detectand monitor pathogenic microorganisms in a test sample, as well as todetect abnormal genes in an individual. Generic amplification systemsare described for clinical use that combine magnetic separationtechniques with ligation/amplification techniques for detecting andmeasuring nucleic acids in a sample. The separation techniques may becombined with most amplification systems, including inter alfa, PCR, LCRand SDA amplification techniques. The present invention further providesalternative amplification systems referred to as ramification-extensionamplification method (RAM) and hybridization signal amplification (HSAM)that are useful in the method of the present invention. The advantagesof the present invention include (1) suitability for clinical laboratorysettings, (2) ability to obtain controlled and consistent(standardizable) results, (3) ability to quantitate nucleic acids in aparticular sample, (4) ability to simultaneously detect and quantitatemultiple target nucleic acids in a test sample, (5) ability tosensitively and efficiently detect nucleic acids in serum samples and insitu, and (6) ability to detect a single mutation in a target. Moreover,the complete protocol of the presently disclosed method may be easilyautomated, making it useful for routine diagnostic testing in a clinicallaboratory setting. With the use of RAM and HSAM, an isothermalamplification can be achieved.

The present invention incorporates magnetic separation, utilizingparamagnetic particles, beads or spheres that have been coated with aligand binding moiety that recognizes and binds to ligand present on anoligonucleotide capture probe, described below, to isolate a targetnucleic acid (DNA or RNA) from a clinical sample in order to facilitateits detection.

Magnetic separation is a system that uses paramagnetic particles orbeads coated with a ligand binding moiety to isolate a target nucleicacid (RNA or DNA) (Lomeli et al. Clin. Chem. 35:1826, 1989) from asample. The principle underscoring this method is one of hybridformation between a capture probe containing a ligand, and a targetnucleic acid through the specific complementary sequence between theprobe and target. Hybridization is carried out in the presence of asuitable chaotropic agent, e.g., guanidine thiocyanate (GnSCN) whichfacilitates the specific binding of the probe to complementary sequencesin the target nucleic acid. The hybrid so formed is then captured on theparamagnetic bead through specific binding of the ligand on the captureprobe to the ligand binding moiety on the bead.

The term “ligand” as used herein refers to any component that has anaffinity for another component termed here as “ligand binding moiety.”The binding of the ligand to the ligand binding moiety forms an affinitypair between the two components. For example, such affinity pairsinclude, inter alia, biotin with avidin/streptavidin, antigens orhaptens with antibodies, heavy metal derivatives with thiogroups,various polynucleotides such as homopolynucleotides as poly dG with polydC, poly dA with poly dT and poly dA with poly U. Any component pairswith strong affinity for each other can be used as the affinity pair,ligand-ligand binding moiety. Suitable affinity pairs are also foundamong ligands and conjugates used in immuno-logical methods. Thepreferred ligand-ligand binding moiety for use in the present inventionis the biotin/streptavidin affinity pair.

In one aspect, the present invention provides for the capture anddetection of a target nucleic acid as depicted in FIG. 1, which providesa schematic depiction of the capture and detection of a target nucleicacid. In the presence of paramagnetic beads or particles (a) coated witha ligand binding moiety (b), the target nucleic acid is hybridizedsimultaneously to a pair of oligonucleotide amplification probes, i.e.,a first nucleotide probe (also referred to as a capture/amplificationprobe) and a second nucleotide probe (also referred to as anamplification probe), designated in FIG. 1 as Capture/Amp-probe-1 (d ande) and Amp-probe-2 (f and g), respectively. The probes may be eitheroligodeoxyribonucleotide or oligoribonucleotide molecules, with thechoice of molecule type depending on the subsequent amplificationmethod. Reference to “probe” herein generally refers to multiple copiesof a probe.

The capture/amplification probe is designed to have a generic 3′nucleotide sequence (d), i.e., it is not specific for the specifictarget nucleic acid being analyzed and thus can be used with a varietyof target nucleic acids. In other words, the 3′ sequence of the firstprobe is not complementary, nor hybridizable, to the nucleotide sequenceof the target nucleic acid. The 5′ portion (e) of thecapture/amplification probe comprises a nucleotide sequence that iscomplementary and hybridizable to a portion of the nucleotide sequenceof the specific target nucleic acid. Preferably, for use with pathogenicmicroorganisms and viruses, the capture/amplification probe issynthesized so that its 3′ generic sequence (d) is the same for allsystems, with the 5′ specific sequence (e) being specificallycomplementary to a target nucleic acid of an individual species orsubspecies of organism or an abnormal gene, e.g., the gene(s)responsible for cystic fibrosis or sickle cell anemia. In certaininstances, it may be desirable that the 5′ specific portion of thecapture/amplification probe be specifically complementary to thenucleotide sequence of a target nucleic acid of a particular strain oforganism. Capture/Amp-probe-1 further contains a ligand (c) at the 3′end of the probe (d), which is recognized by and binds to the ligandbinding moiety (b) coated onto the paramagnetic beads (a).

The second or amplification probe, i.e., Amp-probe-2 in FIG. 1, containsa 3′ sequence (f) that is complementary and hybridizes to a portion ofthe nucleotide sequence of a target nucleic acid immediately adjacent to(but not overlapping) the sequence of the target that hybridizes to the5′ end of Capture/Amp-probe-1. Amp-probe-2 also contains a 5′ genericsequence (g) which is neither complementary nor hybridizable to thetarget nucleic acid, to which may be optionally attached at the 5′ endthereof a label or signal generating moiety (***). Such signalgenerating moieties include, inter alia, radioisotopes, e.g., ³²P or ³H,fluorescent molecules, e.g., fluorescent and chromogenic molecules orenzymes, e.g., peroxidase. Such labels are used for direct detection ofthe target nucleic acid and detects the presence of Amp-probe-2 bound tothe target nucleic acid during the detection step. ³²P is preferred fordetection analysis by radioisotope counting or autoradiography ofelectrophoretic gels. Chromogenic agents are preferred for detectionanalysis, e.g., by an enzyme linked chromogenic assay.

As a result of the affinity of the ligand binding moiety on theparamagnetic beads for the ligand on the capture/amplification probe,target nucleic acid hybridized to the specific 5′ portion of the probeis captured by the paramagnetic beads. In addition, Amp-probe-2, whichhas also hybridized to the target nucleic acid is also captured by theparamagnetic beads.

After capture of the target nucleic acid and the two hybridized probeson the paramagnetic beads, the probes are ligated together (at the sitedepicted by the vertical arrow in FIG. 1) using a ligating agent to forma contiguous single-stranded oligonucleotide molecule, referred toherein as a ligated amplification sequence. The ligating agent may be anenzyme, e.g., a DNA or RNA ligase, or a chemical joining agent, e.g.,cyanogen bromide or a carbodiimide (Sokolova et al., FEBS Lett.232:153-155, 1988). The ligated amplification sequence is hybridized tothe target nucleic acid (either an RNA or DNA) at the region of theligated amplification sequence that is complementary to the targetnucleic acid (e.g., (e) and (f) in FIG. 1).

If a sufficient amount of target nucleic acid (e.g., 10⁶-10′ molecules)is present in the sample, detection of the target nucleic acid can beachieved without any further amplification of the ligation amplificationsequence, e.g., by detecting the presence of the optional signalgenerating moiety at the 5′ end of Amp-probe-2.

If there is insufficient target nucleic acid (e.g., <10⁶ molecules) inthe sample for direct detection, as above, the ligated amplificationsequence formed as described above by the ligation ofCapture/Amp-probe-1 and Amp-probe-2 may be amplified for detection asdescribed below.

Alternatively, a capture/amplification probe, preferably between 70-90nucleotides in length, can be synthesized to contain two ligand moities:one located at the 5′ end and the other located approximately 50nucleotides downstream of the 5′ end. A second circular probe,designated AMP-probe-2, is also synthesized. The linker region of theAMP-probe-2 is complementary to the capture/primer between nucleotide1-50. In the assay system, the capture/amplification probe can bind to aligand binding moiety conjugated to a support matrix, through aligand/ligand binding interaction. Ligands include biotin, antigens,antibodies, heavy metal derivatives and polynucleotides. Ligand bindingmoieties include strepavidin, avidin, antibodies, antigens, thio groups,and polynucleotides. Support matrices include, for example, magneticbeads although other types of supports may be used, including but notlimited to, slides or microtitre plates. The AMP-probe-2 will bind tothe capture/amplification probe through the complementary region. The 3′end of the capture/amplification probe is designed to loop back and bindto 5′ end of the linker region of the AMP-probe-2 and serves as a primerfor extension. Finally, the target can bind to the AMP-probe-2 throughcomplementary regions thereby permitting capture onto a matrix, such asmagnetic beads for example, as depicted in FIG. 24. Ligation will jointhe 3′ and the 5′ end of the AMP-probe-2 and form a covalently linkedcircular probe. Bound probe allows for extensive stringent washes,thereby decreasing the background resulting from non-specific capturing.Extension from the capture/amplification probe along the C-probe willgenerate a multi-unit ssDNA which can then be amplified by either primerextension or RAM by addition of RAM primers as described above. Toincrease assay specificity even further, a double ligation can beperformed, where two probes, each consisting of half of the AMP-probe-2,are used.

In addition, the capture/amplification probe can be designed to bind toan antibody. The AMP-probe-2 as described above will target to thecapture region of the capture/amplification probe (FIG. 25). Afterligation, a primer extension or RAM reaction is carried out as describedabove. Alternatively, one antibody can be attached to acapture/amplification probe and the other antibody can be attached to atarget sequence. In this instance only if both antibodies are bound tothe same antigen will ligation occur. This technique can be used forELISA in a liquid phase RAM reaction or in situ in a solid phase RAMreaction. For the detection purpose, FITC-labeled dUTP or dig-labeleddUTP can be used to detect the RAM products.

Alternately, the ligated amplification sequence can be detected withoutnucleic acid amplification of the ligated sequence by the use of ahybridization signal amplification method (HSAM). HSAM is illustrated inFIG. 10. For HSAM, the target specific nucleic acid probe (e.g.Amp-probe-2) is internally labeled with a ligand. The ligand is amolecule that can be bound to the nucleic acid probe, and can provide abinding partner for a ligand binding molecule that is at least divalent.In a preferred embodiment the ligand is biotin or an antigen, forexample digoxigenin. The nucleic acid probe can be labeled with theligand by methods known in the art. In a preferred embodiment, the probeis labeled with from about 3 to about 10 molecules of ligand, preferablybiotin or digoxigenin. After the capture probe and ligand-labeled targetspecific probe are added to the sample and the resulting complex iswashed as described hereinabove, the ligating agent is added to ligatethe probes as described above. The ligation of the target specific probeto the capture probe results in retention of the target specific probeon the beads. Concurrently or subsequently, an excess of ligand bindingmoiety is added to the reaction. The ligand binding moiety is a moietythat binds to and forms an affinity pair with the ligand. The ligandbinding moiety is at least divalent for the ligand. In a preferredembodiment, the ligand is biotin and the ligand binding moiety isstreptavidin. In another preferred embodiment the ligand is an antigenand the ligand binding molecule is an antibody to the antigen. Additionof ligating agent and ligand binding molecule results in a complexcomprising the target specific probe covalently linked to the captureprobe, with the ligand-labeled target specific probe having ligandbinding molecules bound to the ligand.

A signal probe is then added to the reaction mixture. The signal probeis a generic nucleic acid that is internally labeled with a ligand thatbinds to the ligand binding molecule. In a preferred embodiment, theligand is the same ligand that is used to label the target specificamplification probe. The signal probe has a generic sequence such thatit is not complementary or hybridizable to the target nucleic acid orthe other probes. In a preferred embodiment, the signal probe containsfrom about 30 to about 100 nucleotides and contains from about 3 toabout 10 molecules of ligand.

Addition of the signal probe to the complex in the presence of excessligand binding molecule results in the formation of a large and easilydetectable complex. The size of the complex results from the multiplevalency of the ligand binding molecule. For example, when the ligand inthe target specific amplification probe is biotin, one molecule ofstreptavidin binds per molecule of biotin in the probe. The boundstreptavidin is capable of binding to three additional molecules ofbiotin. When the signal probe is added, the biotin molecules on thesignal probe bind to the available binding sites of the streptavidinbound to the amplification probe. A web-like complex is formed asdepicted schematically in FIG. 10.

Following washing as described hereinabove to remove unbound signalprobe and ligand binding molecules, the complex is then detected.Detection of the complex is indicative of the presence of the targetnucleic acid. The HSAM method thus allows detection of the targetnucleic acid in the absence of nucleic acid amplification.

The complex can be detected by methods known in the art and suitable forthe selected ligand and ligand binding moiety. For example, when theligand binding moiety is streptavidin, it can be detected by immunoassaywith streptavidin antibodies. Alternately, the ligand binding moleculemay be utilized in the present method as a conjugate that is easilydetectable. For example, the ligand may be conjugated with afluorochrome or with an enzyme that is detectable by an enzyme-linkedchromogenic assay, such as alkaline phosphatase or horseradishperoxidase. For example, the ligand binding molecule may be alkalinephosphatase-conjugated streptavidin, which may be detected by additionof a chromogenic alkaline phosphatase substrate, e.g., nitrobluetetrazolium chloride.

The HSAM method may also be used with the circularizable amplificationprobes described hereinbelow. The circularizable amplification probescontain a 3′ and a 5′ region that are complementary and hybridizable toadjacent but not contiguous sequences in the target nucleic acid, and alinker region that is not complementary nor hybridizable to the targetnucleic acid. Upon binding of the circularizable probe to the targetnucleic acid, the 3′ and 5′ regions are juxtaposed. Linkage of the 3′and 5′ regions by addition of a linking agent results in the formationof a closed circular molecule bound to the target nucleic acid. Thetarget/probe complex is then washed extensively to remove unboundprobes.

For HSAM, ligand molecules are incorporated into the linker region ofthe circularizable probe, for example during probe synthesis. The HSAMassay is then performed as described hereinabove and depicted in FIG. 15by adding ligand binding molecules and signal probes to form a largecomplex, washing, and then detecting the complex. Nucleic acid detectionmethods are known to those of ordinary skill in the art and include, forexample, latex agglutination as described by Essers, et al. (1980), J.Clin. Microbiol. 12:641. The use of circularizable probes in conjunctionwith HSAM is particularly useful for in situ hybridization.

HSAM is also useful for detection of an antibody or antigen. Aligand-containing antigen or antibody is used to bind to a correspondingantibody or antigen, respectively. After washing, excess ligand bindingmolecule is then added with ligand-labeled generic nucleic acid probe. Alarge complex is generated and can be detected as described hereinabove.In a preferred embodiment, the ligand is biotin and the ligand bindingmolecule is streptavidin. The use of HSAM to detect an antigen utilizinga biotinylated antibody and biotinylated signal probe is depicted inFIG. 11.

The present methods may be used with routine clinical samples obtainedfor testing purposes by a clinical diagnostic laboratory. Clinicalsamples that may be used in the present methods include, inter alia,whole blood, separated white blood cells, sputum, urine, tissuebiopsies, throat swabbings and the like, i.e., any patient samplenormally sent to a clinical laboratory for analysis.

The present ligation-dependent amplification methods are particularlyuseful for detection of target sequences in formalin fixed, paraffinembedded (FFPE) specimens, and overcomes deficiencies of the prior artmethod of reverse transcription polymerase chain reaction (RT-PCR) fordetection of target RNA sequences in FFPE specimens. RT-PCR has avariable detection sensitivity, presumably because the formation ofRNA-RNA and RNA-protein crosslinks during formalin fixation preventsreverse transcriptase from extending the primers. In the present methodsthe probes can hybridize to the targets despite the crosslinks, reversetranscription is not required, and the probe, rather than the targetsequence, is amplified. Thus the sensitivity of the present methods isnot compromised by the presence of crosslinks. The advantages of thepresent methods relative to RT-PCR are depicted schematically in FIG.12.

With reference to FIG. 2, which provides a general diagrammaticdescription of the magnetic separation and target-dependent detection ofa target nucleic acid in a sample, this aspect of the present methodinvolves the following steps:

(a) The first step is the capture or isolation of a target nucleic acidpresent in the sample being analyzed, e.g., serum. A suitable samplesize for analysis that lends itself well to being performed in amicro-well plate is about 100 μl. The use of micro-well plates foranalysis of samples by the present method facilitates automation of themethod. The sample, containing a suspected pathogenic microorganism orvirus or abnormal gene, is incubated with an equal volume of lysisbuffer, containing a chaotropic agent (i.e., an agent that disruptshydrogen bonds in a compound), a stabilizer and a detergent, whichprovides for the release of any nucleic acids and proteins that arepresent in the sample. For example, a suitable lysis buffer for use inthe present method comprises 2.5-5M guanidine thiocyanate (GnSCN), 10%dextran sulfate, 100 mM EDTA, 200 mM Tris-HCl (pH 8.0) and 0.5% NP-40(Nonidet P-40, a nonionic detergent, N-lauroylsarcosine, Sigma ChemicalCo., St. Louis, Mo.). The concentration of GnSCN, which is a chaotropicagent, in the buffer also has the effect of denaturing proteins andother molecules involved in pathogenicity of the microorganism or virus.This aids in preventing the possibility of any accidental infection thatmay occur during subsequent manipulations of samples containingpathogens.

Paramagnetic particles or beads coated with the ligand binding moietyare added to the sample, either simultaneous with or prior to treatmentwith the lysis buffer. The paramagnetic beads or particles used in thepresent method comprise ferrieoxide particles (generally <1 um indiameter) that possess highly convoluted surfaces coated with siliconhydrides. The ligand binding moiety is covalently linked to the siliconhydrides. The paramagnetic particles or beads are not magneticthemselves and do not aggregate together. However, when placed in amagnetic field, they are attracted to the magnetic source. Accordingly,the paramagnetic particles or beads, together with anything bound tothem, may be separated from other components of a mixture by placing thereaction vessel in the presence of a strong magnetic field provided by amagnetic separation device. Such devices are commercially available,e.g., from Promega Corporation or Stratagene, Inc.

Suitable paramagnetic beads for use in the present method are thosecoated with streptavidin, which binds to biotin. Such beads arecommercially available from several sources, e.g., StreptavidinMagneSphere® paramagnetic particles obtainable from Promega Corporationand Streptavidin-Magnetic Beads (catalog #MB002) obtainable fromAmerican Qualex, La Mirada, Calif.

Subsequently, a pair of oligonucleotide amplification probes, asdescribed above, is added to the lysed sample and paramagnetic beads. Ina variation, the probes and paramagnetic beads may be added at the sametime. As described above, the two oligonucleotide probes are a firstprobe or capture/amplification probe (designated Capture/Amp-probe-1 inFIG. 1) containing a ligand at its 3′ end and a second probe oramplification probe (designated Amp-probe-2 in FIG. 1). For use withstreptavidin-coated paramagnetic beads, the first probe is preferably a3′-biotinylated capture/amplification probe.

The probes may be synthesized from nucleoside triphosphates by knownautomated oligonucleotide synthetic techniques, e.g., via standardphosphoramidite technology utilizing a nucleic acid synthesizer. Suchsynthesizers are available, e.g., from Applied Biosystems, Inc. (FosterCity, Calif.).

Each of the oligonucleotide probes are about 40-200 nucleotides inlength, preferably about 50-100 nucleotides in length, which, afterligation of the probes, provides a ligated amplification sequence ofabout 80-400, preferably 100-200, nucleotides in length, which issuitable for amplification via PCR, Qβ replicase or SDA reactions.

The target nucleic acid specific portions of the probes, i.e., the 5′end of the first capture/amplification probe and the 3′ end of thesecond amplification probe complementary to the nucleotide sequence ofthe target nucleic acid, are each approximately 15-60 nucleotides inlength, preferably about 18-35 nucleotides, which provides a sufficientlength for adequate hybridization of the probes to the target nucleicacid.

With regard to the generic portions of the probes, i.e., the 3′ end ofthe capture/amplification probe and the 5′ end of the amplificationprobe, which are not complementary to the target nucleic acid, thefollowing considerations, inter glia, apply:

(1) The generic nucleotide sequence of an oligodeoxynucleotidecapture/amplification probe comprises at least one and, preferably twoto four, restriction endonuclease recognition sequences(s) of about sixnucleotides in length, which can be utilized, if desired, to cleave theligated amplification sequence from the paramagnetic beads by specificrestriction endonucleases, as discussed below. Preferred restrictionsites include, inter alia, EcoRI (GAATTC), SmaI (CCCGGG) and HindIII(AAGCTT).

(2) The generic nucleotide sequence comprises a G-C rich region which,upon hybridization to a primer, as discussed below, provides a morestable duplex molecule, e.g., one which requires a higher temperature todenature. Ligated amplification sequences having G-C rich genericportions of the capture/amplification and amplification probes may beamplified using a two temperature PCR reaction, wherein primerhybridization and extension may both be carried out at a temperature ofabout 60-65° C. (as opposed to hybridizing at 37° C., normally used forPCR amplification) and denaturation at a temperature of about 92° C., asdiscussed below. The use of a two temperature reaction reduces thelength of each PCR amplification cycle and results in a shorter assaytime.

Following incubation of the probes, magnetic beads and target nucleicacid in the lysis buffer for about 30-60 minutes, at a temperature ofabout 37° C., a ternary complex comprising the target nucleic acid andhybridized probes is formed, which is bound to the paramagnetic beadsthrough the binding of the ligand (e.g., biotin) on thecapture/amplification probe to the ligand binding moiety (e.g.,streptavidin) on the paramagnetic beads. The method is carved out asfollows:

(a) The complex comprising target nucleic acid-probes-beads is thenseparated from the lysis buffer by means of a magnetic field generatedby a magnetic device, which attracts the beads. The magnetic field isused to hold the complex to the walls of the reaction vessel, e.g., amicro-well plate or a microtube, thereby allowing for the lysis bufferand any unbound reactants to be removed, e.g., by decanting, without anyappreciable loss of target nucleic acid or hybridized probes. Thecomplex is then washed 2-3 times in the presence of the magnetic fieldwith a buffer that contains a chaotropic agent and detergent in amountsthat will not dissociate the complex. A suitable washing buffer for usein the present method comprises about 1.0-1.5M GnSCN, 10 mM EDTA, 100 mMTris-HCl (pH 8.0) and 0.5% NP-40 (Nonidet P-40, nonionic detergent,Sigma Chemical Co., St. Louis, Mo.). Other nonionic detergents, e.g.,Triton X-100, may also be used. The buffer wash removes unboundproteins, nucleic acids and probes that may interfere with subsequentsteps. The washed complex may be then washed with a solution of KCl toremove the GnSCN and detergent and to preserve the complex. A suitableconcentration of KCl is about 100 to 500 mM KCl. Alternatively, the KClwash step may be omitted in favor of two washes with ligase buffer.

(b) If the probes are to be ligated together, the next step in thepresent method involves treating the complex from step (a) with aligating agent that will join the two probes. The ligating agent may bean enzyme, e.g., DNA or RNA ligase, or a chemical agent, e.g., cyanogenbromide or a carbodiimide. This serves to join the 5′ end of the firstoligonucleotide probe to the 3′ end of the second oligonucleotide probe(capture/amplification probe and amplification probe, respectively) toform a contiguous functional single-stranded oligonucleotide molecule,referred to herein as a ligated amplification sequence. The presence ofthe ligated amplification sequence detected, (via the signal generatingmoiety at the 5′ end of Amp-probe-2), indirectly indicates the presenceof target nucleic acid in the sample. Alternatively, the ligatedamplification sequence serves as the template for any of variousamplification systems, such as PCR or SDA. Any of the first and secondprobes which remain unligated after treatment are not amplified insubsequent steps in the method. Capture/amplification and amplificationoligodeoxynucleotide probes may be ligated using a suitable ligatingagent, such as a DNA or RNA ligase. Alternatively, the ligating agentmay be a chemical, such as cyanogen bromide or a carbodiimide (Sokolovaet al., FEBS Lett. 232:153-155, 1988). Preferred DNA ligases include T₄DNA ligase and the thermostable Taq DNA ligase, with the latter beingmost preferable, for probes being subjected to amplification using PCRtechniques. The advantage of using the Taq DNA ligase is that it isactive at elevated temperatures (65-72° C.). Joining the oligonucleotideprobes at such elevated temperatures decreases non-specific ligation.Preferably, the ligation step is carried out for 30-60 minutes at anelevated temperature (about 65-72° C.), after which time any unligatedsecond amplification probe (Amp-probe-2 in FIG. 1) may be, optionally,removed under denaturing conditions.

Denaturation is performed after the ligation step by adding TE Buffer(10 mM Tris-HCl pH 7.5, 0.1 mM EDTA) to the mixture. The temperature ofthe mixture is then raised to about 92-95° C. for about 1-5 minutes todenature the hybridized nucleic acid. This treatment separates thetarget nucleic acid (and unligated Amp-probe-2) from the hybridizedligated amplification sequences, which remains bound to the paramagneticbeads. In the presence of a magnetic field, as above, the bound ligatedamplification sequence is washed with TE Buffer at elevated temperatureto remove denatured target nucleic acid and any unligated Amp-probe-2and resuspended in TE Buffer for further analysis.

(c) The third step in the process is detection of the ligatedamplification sequence, which indicates the presence of the targetnucleic acid in the original test sample. This may be performed directlyif sufficient target nucleic acid (about 10⁶-10⁷ molecules) is presentin the sample or following amplification of the ligated amplificationsequence, using one of the various amplification techniques, e.g., PCRor SDA. For example, direct detection may be used to detect HIV-1 RNA ina serum sample from an acutely infected AIDS patient. Such a serumsample is believed to contain about 10⁶ copies of the viral RNA/ml.

For direct detection, an oligonucleotide detection probe ofapproximately 10-15 nucleotides in length, prepared by automativesynthesis as described above to be complementary to the 5′ end of theAmp-probe-2 portion of the ligated amplification sequence, may be addedto the ligated amplification sequence attached to the paramagneticbeads. The detection probe, which is labeled with a signal generatingmoiety, e.g., a radioisotope, a chromogenic agent or a fluorescentagent, is incubated with the complex for a period of time and underconditions sufficient to allow the detection probe to hybridize to theligated amplification sequence. The incubation time can range from about1-60 minutes and may be carried out at a temperature of about 4-60° C.Preferably, when the label is a fluorogenic agent, the incubationtemperature is about 4° C.; a chromogenic agent, about 37° C.; and aradioisotope, about 37°-60° C. Preferred signal generating moietiesinclude, inter alia, ³²P (radioisotope), peroxidase (chromogenic) andfluorescein, acridine or ethidium (fluorescent).

Alternatively, for direct detection, as discussed above, the Amp-probe-2itself may be optionally labeled at its 5′ end with a signal generatingmoiety, e.g., ³²P. The signal generating moiety will then beincorporated into the ligated amplification sequence following ligationof the Capture/Amp-probe-1 and Amp-probe-2. Thus, direct detection ofthe ligated amplification sequence, to indicate the presence of thetarget nucleic acid, can be carried out immediately following ligationand washing.

Any suitable technique for detecting the signal generating moietydirectly on the ligated amplification probe or hybridized thereto viathe detection primer may be utilized. Such techniques includescintillation counting (for ³²P) and chromogenic or fluorogenicdetection methods as known in the art. For example, suitable detectionmethods may be found, inter alia, in Sambrook et al., MolecularCloning—A Laboratory Manual, 2d Edit., Cold Spring Harbor Laboratory,1989, in Methods in Enzymology, Volume 152, Academic Press (1987) or Wuet al., Recombinant DNA Methodology, Academic Press (1989).

If an insufficient amount of target nucleic acid is present in theoriginal sample (<10⁶ molecules), an amplification system is used toamplify the ligated amplification sequence for detection.

For example, if the probes used in the present method areoligodeoxyribonucleotide molecules, PCR methodology can be employed toamplify the ligated amplification sequence, using known techniques (see,e.g., PCR Technology, H. A. Erlich, ed., Stockton Press, 1989, Sambrooket al., Molecular Cloning—A Laboratory Manual, 2d Edit., Cold SpringHarbor Laboratory, 1989. When using PCR for amplification, two primersare employed, the first of the primers being complementary to thegeneric 3′ end of Capture/Amp-probe-1 region of the ligatedamplification sequence and the second primer corresponding in sequenceto the generic 5′ end of Amp-probe-2 portion of the ligatedamplification sequence. These primers, like the sequences of the probesto which they bind, are designed to be generic and may be used in allassays, irrespective of the sequence of the target nucleic acid. Becausethe first primer is designed to anneal to the generic sequence at the 3′end of the ligated amplification sequence and the second primercorresponds in sequence to the generic sequence at the 5′ end of theligated amplification sequence, generic primers may be utilized toamplify any ligated amplification sequence.

Alternatively, multiple primers, designed to be complementary to thegeneric 3′ end of the Capture/AMP-probe-1 region of the ligatedamplification sequence and the generic 5′ end of the AMP-probe-2 portionof the ligated amplification sequence may be used to amplify ligatedamplification sequence together with the sequence between both ends. Asdemonstrated in the working examples described herein, increasing thenumber of primers was demonstrated to significantly increase theamplification efficiency thereby increasing the sensitivity of DNAdetection.

A generic pair of PCR oligonucleotide primers for use in the presentmethod may be synthesized from nucleoside triphosphates by knownautomated synthetic techniques, as discussed above for synthesis of theoligonucleotide probes. The primers may be 10-60 nucleotides in length.Preferably the oligonucleotide primers are about 18-35 nucleotides inlength, with lengths of 12-21 nucleotides being most preferred. The pairof primers are designated to be complementary to the generic portions ofthe first capture/amplification probe and second amplification probe,respectively and thus have high G-C content. It is also preferred thatthe primers are designed so that they do not have any secondarystructure, i.e., each primer contains no complementary region withinitself that could lead to self annealing.

The high G-C content of the generic PCR primers and the generic portionsof the ligated amplification sequence permits performing the PCRreaction at two temperatures, rather than the usual three temperaturemethod. Generally, in the three temperature method, each cycle ofamplification is carried out as follows:

Annealing of the primers to the ligated amplification sequence iscarried out at about 37-50° C.; extension of the primer sequence by Taqpolymerase in the presence of nucleoside triphosphates is carried out atabout 70-75° C.; and the denaturing step to release the extended primeris carried out at about 90-95° C. In the two temperature PCR technique,the annealing and extension steps may both be carried at about 60-65°C., thus reducing the length of each amplification cycle and resultingin a shorter assay time.

For example, a suitable three temperature PCR amplification (as providedin Saiki et al., Science 239:487-491, 1988) may be carried out asfollows:

Polymerase chain reactions (PCR) are carved out in about 25-50 μlsamples containing 0.01 to 1.0 ng of template ligated amplificationsequence, 10 to 100 pmol of each generic primer, 1.5 units of Taq DNApolymerase (Promega Corp.), 0.2 mM dATP, 0.2 mM dCTP, 0.2 mM dGTP, 0.2mM dTTP, 15 mM MgCl₂, 10 mM Tris-HCl (pH 9.0), 50 mM KCl, 1 μl/mlgelatin, and 10 μl/ml Triton X-100 (Saiki, 1988). Reactions areincubated at 94° C. for 1 minute, about 37 to 55° C. for 2 minutes(depending on the identity of the primers), and about 72° C. for 3minutes and repeated for 30-40, preferably 35, cycles. A 4 μl-aliquot ofeach reaction is analyzed by electrophoresis through a 2% agarose geland the DNA products in the sample are visualized by staining the gelwith ethidium-bromide.

The two temperature PCR technique, as discussed above, differs from theabove only in carrying out the annealing/extension steps at a singletemperature, e.g., about 60-65° C. for about 5 minutes, rather than attwo temperatures.

Also, with reference to FIG. 2, quantitative detection of the targetnucleic acid using a competitive PCR assay may also be carried out. Forsuch quantitative detection, a oligodeoxyribonucleotide releasingprimer, synthesized generally as described above, is added to theparamagnetic bead-bound ligated amplification sequence. The releasingprimer, may or may not be but, preferably, will be the same as the firstPCR primer discussed above. The releasing primer is designed tohybridize to the generic 3′ end of the Capture/Amp-probe-1 portion ofthe ligated amplification sequence, which, as discussed above, comprisesa nucleotide sequence recognized by at least one, and preferablytwo-four, restriction endonucleases to form at least one, and preferablytwo-four, double-stranded restriction enzyme cleavage site, e.g., aEcoRI, SmaI and/or HindIII site(s).

In this regard, as noted above, for use in a quantitative PCRamplification and detection system, it is important that theCapture/Amp-probe-1 be synthesized with at least one, and preferably twoto four nucleotide sequences recognized by a restriction enzyme locatedat the 3′ end of the probe. This provides the nucleotide sequences towhich the releasing primer binds to form double-stranded restrictionenzyme cleavage site(s).

After ligating the first and second probes to form the ligatedamplification sequence, the releasing primer is hybridized to theligated amplification sequence. At least one restriction enzyme, e.g.,EcoRI, SmaI and/or HindIII, is then added to the hybridized primer andligated amplification sequence. The ligated amplification sequence isreleased from the beads by cleavage at the restriction enzyme, e.g.,EcoRI site.

Following its release from the beads, the ligated amplification sequenceis serially diluted and then quantitatively amplified via the DNA Taqpolymerase using a suitable PCR amplification technique, as describedabove.

Quantitation of the original target nucleic acid in the sample may beperformed by a competitive PCR method to quantitatively amplify theligated amplification sequence, as provided, e.g., in Sambrook et al.,Molecular Cloning—A Laboratory Manual, 2d Edit., Cold Spring HarborLaboratory, 1989.

In general, the method involves co-amplification of two templates: theligated amplification sequence and a control (e.g., the generic portionsof the ligated amplification sequence or the generic portions that haveinterposed thereto a nucleotide sequence unrelated to the sequence ofthe target nucleic acid) added in known amounts to a series ofamplification reactions. While the control and ligated amplificationsequence are amplified by the same pair of generic PCR primers, thecontrol template is distinguishable from the ligated amplificationsequence, e.g., by being different in size. Because the control andligated amplification sequence templates are present in the sameamplification reaction and use the same primers, the effect of a numberof variables which can effect the efficiency of the amplificationreaction is essentially nullified. Such variables included, inter alia:(1) quality and concentration of reagents (Tag DNA polymerase, primers,templates, dNTP's), (2) conditions used for denaturation, annealing andprimer extension, (3) rate of change of reaction temperature and (4)priming efficiency of the oligonucleotide primers. The relative amountsof the two amplified products—i.e., ligated amplification sequence andcontrol template—reflect the relative concentrations of the startingtemplates.

The quantitative PCR method may be generally carried out as follows:

A control template, e.g., a DNA sequence corresponding to nanovariantRNA, a naturally occurring template of Qβ replicase (Schaffner et al.,J. Mol. Biol. 117:877-907, 1977) is synthesized by automatedoligonucleotide synthesis and its concentration determined, e.g., byspectrophotometry or by ethidium-bromide mediated fluorescence.

A series of tenfold dilutions (in TE Buffer) containing from 10 ng/ml to1 fg/ml of the control template is made and stored at −70° C. until use.

A series of PCR amplification reactions of the free ligatedamplification sequence is set up. In addition to the usual PCRingredients, the reactions also contain about 10μ/reaction of thetenfold dilutions of the control template and about 10 μCi/reaction ofα-³²P] dCTP(Sp.act. 3000 Ci/mmole).

PCR amplification reactions are carried out for a desired number ofcycles, e.g., 30-40.

The reaction products may then be subject to agarose gel electrophoresisand autoradiography to separate the two amplified products (of differentsizes). The amplified bands of the control and ligated amplificationsequence are recovered from the gel using suitable techniques andradioactivity present in each band is determined by counting in ascintillation counter. The relative amounts of the two products arecalculated based on the amount of radioactivity in each band. The amountof radioactivity in the two samples must be corrected for thedifferences in molecular weights of the two products.

The reactions may be repeated using a narrower range of concentration ofcontrol template to better estimate the concentration of ligatedamplification sequence.

In another aspect of the invention, more than the two probes i.e. asingle capture/amplification probe, and a single amplification probe maybe utilized. For example one or more capture/amplification probes, andone or more amplification probes, may be employed in the detection andcapture of the target nucleic acid, and optional amplification of thetarget sequences, as shown schematically in FIGS. 4 and 5. According tothis aspect of the present invention, the capture/amplification probesmay have a 3′ sequence complementary to the target nucleic acid and abiotin moiety at the 5′ terminus that is capable of interacting with thestreptavidin coated paramagnetic beads. Alternatively, thecapture/amplification probes may have a 5′ sequence complementary to thetarget nucleic acid and a biotin moiety at the 3′ terminus.

Further, according to this aspect of the present invention, one or moreamplification probes are utilized such that each probe containssequences that are specifically complementary to and hybridizable withthe target nucleic acid. For example, the 5′ end of one amplificationprobe, e.g. Amp-probe-2 (HCV A) in FIG. 4, contains a sequencecomplementary to a distinct portion in the target nucleic acid. The 3′end of the second amplification probe e.g. Amp-probe-2A (HCV A) in FIG.4, contains a specific sequence complementary to a region of the targetnucleic acid that is immediately adjacent to that portion of the targethybridizable to the first amplification probe. The capture/amplificationprobes and the pair of amplification probes hybridize with the targetnucleic acid in the presence of GnSCN as described above. This complexso formed is bound to streptavidin-coated paramagnetic beads by means ofa biotin moiety on the capture/amplification probes and the complexseparated from unreacted components by means of magnetic separation asabove. Next, the amplification probes may be linked, for example, by aligase enzyme. This produces a ligated amplification sequence thatserves as a template for Taq DNA polymerase during amplificationreaction by PCR.

In a particular aspect of the invention, two or morecapture/amplification probes and two pairs of amplification probes areutilized for the detection of the target nucleic acid.

The use of multiple capture/amplification probes affords even bettercapture efficiency, permitting the capture of multiple targets withgeneric capture probes. This is especially desirable for multiplex PCRreactions where multiple targets within a single reaction may bedetected.

For example, a capture/amplification probe for use in the present methodmay be designed to bind to the poly-A tail region of cellular mRNA,whereby all such mRNA can be isolated by a single capture-and-wash step.Subsequent PCR amplification may be designed to detect and amplifyspecific target pathogen or disease gene sequences from such an mRNApool. Such genes may include, inter alia, the gene encoding the cysticfibrosis transmembrane regulator protein (CFTR) or hemoglobins or otherproteins involved in genetic diseases.

In still another aspect of the invention, the multiplecapture/amplification probes may target, for example, all strains of aparticular pathogen, e.g. The Hepatitis C Virus (HCV), and amplificationprobes may be tailored to detect and further identify individual HCVgenotypes of the pathogen (e.g. HCV).

In a further embodiment, two capture/amplification probes are utilized.e.g. as depicted in FIG. 4. This provides a total specific sequence ofthe capture/amplification probes complementary and hybridizable to thetarget nucleic acid that can be twice as long as that of a singlecapture/amplification probe, thereby affording an even higher captureefficiency.

The pair of capture/amplification probes, e.g. as shown in FIG. 4, mayeach have a 3′ sequence complementary to the target nucleic acid, and abiotin moiety at its 5′ terminus capable of interacting withstreptavidin coated paramagnetic beads. Alternatively, the pair ofcapture/amplification probes may each have a 5′ sequence complementaryto the target nucleic acid, and a biotin moiety at its 3′ terminuscapable of interacting with streptavidin coated paramagnetic beads.

Further, the present method in which the ligated target probe isamplified by PCR permits the detection of multiple targets in a singlereaction, as illustrated in FIG. 13 and designated as multiplex LD-PCR.In the prior art methods of PCR amplification of a target nucleic acid,attempts to detect multiple targets with multiple primer pairs in asingle reaction vessel have been limited by varying primer efficienciesand competition among primer pairs. In contrast, in the present methodeach capture/amplification probe has a target specific region and ageneric region. In multiplex LD-PCR according to the present invention,the generic regions to which the PCR primers bind may be common to allcapture/amplification probes. Thus multiple pairs ofcapture/amplification probes having specificity for multiple targets maybe used, but only one pair of generic PCR primers are needed to amplifythe ligated capture/amplification probes. By varying the length of thetarget specific regions of the capture/amplification probes, amplifiedPCR products corresponding to a particular target can be identified bysize.

The PCR products may also be identified by an enzyme-linkedimmunosorbent assay (ELISA). The PCR product may be labeled duringamplification with an antigen, for example digoxigenin. The labeled PCRproduct is then captured on a microtiter plate having thereon a nucleicacid probe that hybridizes to the target specific region of theamplification probe, which region is present in the amplified product.The labeled captured product may then be detected by adding an enzymeconjugated antibody against the antigen label, for example horseradishperoxidase anti-digoxigenin antibody, and a color indicator to each wellof the microtiter plate. The optical density of each well provides ameasure of the amount of PCR product, which in turn indicates thepresence of the target nucleic acid in the original sample.

In still further embodiments, the present invention may utilize a singleamplifiable “full length probe” and one or more capture/amplificationprobes as shown in FIG. 6. Further, the hybridized nucleic acid duplex,comprising of the target nucleic acid, for example, HCV RNA, and thecapture/amplification probes and full length amplification probes, alsoreferred to as amplification sequences, can be released from themagnetic beads by treating the hybridized duplex molecule with RNAase H.Alternatively, the hybridized duplex, comprising of the target nucleicacid, e.g. DNA, and the capture/amplification probes and full lengthamplification probes, can be released from the magnetic beads bytreating the hybridized duplex molecule with appropriate restrictionenzymes, as described above.

When a full length amplification probe is employed to detect a targetnucleic acid sequence, the probe may be utilized in amplificationreactions such as PCR, without having to use the ligation step describedabove. This latter approach, in particular, simplifies the assay and isespecially useful when at least 10⁴ target nucleic acid molecules areavailable in the testing sample, so that the chances of non-specificbinding in a ligation independent detection reaction are reduced. Inmost clinical detection assays, the target nucleic acid (such as apathogen), is present at >10⁵ molecules/ml. of sample, and thus would beamenable to detection and amplification by this method.

A still further aspect of the present invention utilizes one or morecapture/amplification probes, each containing a biotin moiety, and asingle amplification probe, also referred to as an amplificationsequence, that hybridizes to the target nucleic acid and circularizesupon ligation of its free termini, as shown in FIG. 7. The amplificationprobe may be designed so that complementary regions (see e.g. The regionshown in bold in FIG. 7) of the probe that are hybridizable to thetarget nucleic acid sequence are located at each end of the probe (asdescribed in Nilsson et al., 1994, Science 265:2085-2088). When theprobe hybridizes with the target, its termini are placed adjacent toeach other, resulting in the formation of a closed circular moleculeupon ligation with a linking agent such as a ligase enzyme. Thiscircular molecule may then serve as a template during an amplificationstep, e.g. PCR, using primers such as those depicted in FIG. 7. Thecircular molecule may also be amplified by RAM, as describedhereinbelow, or detected by a modified HSAM assay, as describedhereinbelow.

For example, the probe, described above, can be used to detect differentgenotypes of a pathogen, e.g. different genotypes of HCV from serumspecimens. Genotype specific probes can be designed, based on publishedHCV sequences (Stuyver et al., 1993, J. Gen. Virol. 74: 1093-1102), suchthat a mutation in the target nucleic acid is detectable since such amutation would interfere with (1) proper hybridization of the probe tothe target nucleic acid and (2) subsequent ligation of the probe into acircular molecule. Because of the nature of the circularized probe, asdiscussed below, unligated probes may be removed under stringent washingconditions.

The single, full length, ligation-dependent circularizable probe, asutilized in the method, affords greater efficiency of the detection andamplification of the target nucleic acid sequence. Due to the helicalnature of double-stranded nucleic acid molecules, circularized probesare wound around the target nucleic acid strand. As a result of theligation step, the probe may be covalently bound to the target moleculeby means of catenation. This results in immobilization of the probe onthe target molecule, forming a hybrid molecule that is substantiallyresistant to stringent washing conditions. This results in significantreduction of non-specific signals during the assay, lower backgroundnoise and an increase in the specificity of the assay.

Another embodiment of the present invention provides a method ofreducing carryover contamination and background in amplification methodsutilizing circular probes. The present ligation-dependent amplificationmethods, unlike conventional amplification methods, involveamplification of the ligated probe(s) rather than the target nucleicacid. When the ligated probe is a closed circular molecule, it has nofree ends susceptible to exonuclease digestion. After probe ligation,i.e. circularization, treatment of the reaction mixture with anexonuclease provides a “clean-up” step and thus reduces background andcarryover contamination by digesting unligated probes or linear DNAfragments but not closed circular molecules. The covalently linkedcircular molecules remain intact for subsequent amplification anddetection. In conventional PCR, the use of exonuclease to eliminatesingle stranded primers or carryover DNA fragments poses the risk thattarget nucleic acid will also be degraded. The present invention doesnot suffer this risk because target nucleic acid is not amplified. In apreferred embodiment, the exonuclease is exonuclease III, exonucleaseVII, mung bean nuclease or nuclease BAL-31. Exonuclease is added to thereaction after ligation and prior to amplification, and incubated, forexample at 37° C. for thirty minutes.

It is further contemplated to use multiple probes which can be ligatedto form a single covalently closed circular probe. For example, a firstprobe is selected to hybridize to a region of the target. A second probeis selected such that its 3′ and 5′ termini hybridize to regions of thetarget that are adjacent but not contiguous with the 5′ and 3′ terminiof the first probe. Two ligation events are then required to provide acovalently closed circular probe. By using two ligases, e.g. anenzymatic and a chemical ligase, to covalently close the probe, theorder of the ligations can be controlled. This embodiment isparticularly useful to identify two nearby mutations in a single target.

The circularized probe can also be amplified and detected by thegeneration of a large polymer. The polymer is generated through therolling circle extension of primer 1 along the circularized probe anddisplacement of downstream sequence. This step produces a singlestranded DNA containing multiple units which serves as a template forsubsequent PCR, as depicted in FIGS. 9 and 16. As shown therein, primer2 can bind to the single stranded DNA polymer and extend simultaneously,resulting in displacement of downstream primers by upstream primers. Byusing both primer-extension/displacement and PCR, more detectableproduct is produced with the same number of cycles.

The circularized probe may also be detected by a modification of theHSAM assay. In this method, depicted in FIG. 14, the circularizableamplification probe contains, as described hereinabove, 3′- and 5′regions that are complementary to adjacent regions of the target nucleicacid. The circularizable probes further contain a non-complementary, orgeneric linker region. In the present signal amplification method, thelinker region of the circularizable probe contains at least one pair ofadjacent regions that are complementary to the 3′ and 5′ regions of afirst generic circularizable signal probe (CS-probe). The first CS-probecontains, in its 3′ and 5′ regions, sequences that are complementary tothe adjacent regions of the linker region of the circularizableamplification probe. Binding of the circularizable amplification probeto the target nucleic acid, followed by ligation, results in acovalently linked circular probe having a region in the linker availablefor binding to the 3′ and 5′ ends of a first CS-probe. The addition ofthe first CS-probe results in binding of its 3′ and 5′ regions to thecomplementary regions of the linker of the circular amplification probe.The 3′ and 5′ regions of the CS-probe are joined by the ligating agentto form a closed circular CS-probe bound to the closed circularamplification probe. The first CS-probe further contains a linker regioncontaining at least one pair of adjacent contiguous regions designed tobe complementary to the 3′ and 5′ regions of a second CS-probe.

The second CS-probe contains, in its 3′ and 5′ regions, sequences thatare complementary to the adjacent regions of the linker region of thefirst CS-probe. The addition of the second CS-probe results in bindingof its 3′ and 5′ regions to the complementary regions of the linker ofthe first CS-probe. The 3′ and 5′ regions of the second CS-probe arejoined by the ligating agent to form a closed circular CS-probe, whichis in turn bound to the closed circular amplification probe.

By performing the above-described method with a multiplicity ofCS-probes having multiple pairs of complementary regions, a largecluster of chained molecules is formed on the target nucleic acid. In apreferred embodiment, three CS-probes are utilized. In addition to the3′ and 5′ regions, each of the CS-probes has one pair of complementaryregions that are complementary to the 3′ and 5′ regions of a secondCS-probe, and another pair of complementary regions that arecomplementary to the 3′ and 5′ regions of the third CS-probe. Byutilizing these “trivalent” CS-probes in the method of the invention, acluster of chained molecules as depicted in FIG. 14 is produced.

Following extensive washing to remove non-specific chain reactions thatare unlinked to the target, the target nucleic acid is then detected bydetecting the cluster of chained molecules. The chained molecules can beeasily detected by digesting the complex with a restriction endonucleasefor which the recognition sequence has been uniquely incorporated intothe linker region of each CS-probe. Restriction endonuclease digestionresults in a linearized monomer that can be visualized on apolyacrylamide gel. Other methods of detection can be effected byincorporating a detectable molecule into the CS-probe, for exampledigoxigenin, biotin, or a fluorescent molecule, and detecting withanti-digoxinin, streptavidin, or fluorescence detection. Latexagglutination, as described for example by Essers et al. (1980) J. Clin.Microbiol. 12, 641, may also be used. Such nucleic acid detectionmethods are known to one of ordinary skill in the art.

Moreover, in a special application, the amplification probes and/oramplification sequences as described above, can be used for in situLD-PCR assays. In situ PCR may be utilized for the direct localizationand visualization of target viral nucleic acids and may be furtheruseful in correlating viral infection with histopathological finding.

Current methods assaying for target viral RNA sequences have utilized RTPCR techniques for this purpose (Nuovo et al., 1993, Am. J. Surg.Pathol. 17(7):683-690). In this method cDNA, obtained from target viralRNA by in situ reverse transcription, is amplified by the PCR method.Subsequent intracellular localization of the amplified cDNA can beaccomplished by in situ hybridization of the amplified cDNA with alabeled probe or by the incorporation of labeled nucleotide into the DNAduring the amplification reaction.

However, the RT PCR method suffers drawbacks which are overcome by thepresent invention. For example, various tissue fixatives used to treatsample tissues effect the crosslinking of cellular nucleic acids andproteins, e.g. protein-RNA and RNA-RNA complexes and hinder reversetranscription, a key step in RT-PCR. Moreover, secondary structures intarget RNA may also interfere with reverse transcription. Further, theapplication of multiplex PCR to RT PCR for the detection of multipletarget sequences in a single cell can present significant problems dueto the different efficiencies of each primer pair.

The method of the present invention utilizes one or more amplificationprobes and/or amplification sequences, as described above, and theLD-PCR technique to locate and detect in situ target nucleic acid, whichoffers certain advantages over the RT-PCR method. First, sincehybridization of the probe to target nucleic acid and subsequentamplification of the probe sequences eliminates the reversetranscription step of the RT-PCR method, the secondary structure of thetarget RNA does not affect the outcome of the assay. Moreover, thecrosslinking of target nucleic acids and cellular proteins due to tissuefixatives, as discussed above, does not interfere with the amplificationof probe sequences since there is no primer extension of the target RNAas in the RT-PCR method.

In particular, amplification probes according to the present inventionmay be designed such that there are common primer-binding sequenceswithin probes detecting different genotypic variants of a targetpathogen. This enables the assay to detect multiple targets in a singlesample. For example, and not by way of limitation, the assay may utilizetwo or more amplification probes according to this method to detect HCVRNA and β-actin RNA, whereby the β-actin probe serves as an internalcontrol for the assay.

Furthermore, the primer-binding sequences in the probe may be designedto (1) minimize non-specific primer oligomerization and (2) achievesuperior primer-binding and increased yield of PCR products, therebyincreasing sensitivity of the assay.

Since the amplification probe circularizes after binding to targetnucleic acid to become a circular molecule, multimeric products may begenerated during polymerization, so that amplification products areeasily detectable, as described above, as shown in FIGS. 9 and 16.

An in situ LD-PCR assay to detect target nucleic acids in histologicalspecimens according to the present invention utilizes a ligationdependent full length amplification probe, and involves the followingsteps:

Sample tissue, e.g., liver, that may be frozen, or formalin-fixed andembedded in paraffin, is sectioned and placed on silane-coated slides.The sections may be washed with xylene and ethanol to remove theparaffin. The sections may then be treated with a proteolytic enzyme,such as trypsin, to increase membrane permeability. The sections may befurther treated with RNAase-free DNAase to eliminate cellular DNA.

An amplification probe may be suspended in a suitable buffer and addedto the sample sections on the slide and allowed to hybridize with thetarget sequences. Preferably, the probe may dissolved in 2×SSC with 20%formamide, added to the slide, and the mixture incubated for 2 hours at37° C. for hybridization to occur. The slide may be washed once with2×SSC and twice with 1× ligase buffer before DIVA ligase may be appliedto the sample. Preferably, IU/20 μl of the ligase enzyme may be added toeach slide, and the mixture incubated at 37° C. for 2 hours to allowcircularization of the probe. The slide may be washed with 0.2×SSC (highstringency buffer) and 1×PCR buffer to remove unligated probes beforethe next step of amplification by PCR. The PCR reaction mixture,containing the amplification primers and one or more labeled nucleotidesis now added to the sample on the slide for the amplification of thetarget sequences. The label on the nucleotide(s) may be any signalgenerating moiety, including, inter alia, radioisotopes, e.g., ³²P or³H, fluorescent molecules, e.g., fluorescenn and chromogenic moleculesor enzymes, e.g., peroxidase, as described earlier. Chromogenic agentsare preferred for detection analysis, e.g., by an enzyme linkedchromogenic assay.

In a still preferred aspect, digoxinin-labeled nucleotides are utilized.In such cases the PCR product, tagged with digoxinin-labeled nucleotidesis detectable when incubated with an antidigoxinin antibody-alkalinephosphatase conjugate. The alkaline phosphatase-based colorimetricdetection utilizes nitroblue tetrazolium, which, in the presence of5-Bromo-4-chloro-3-indolylphosphate, yields a purple-blue precipitate atthe amplification site of the probe.

In one aspect of the present invention, the ligation and the PCRamplification step of the in situ LD-PCR detection method can be carvedout simultaneously and at a higher temperature, by using a thermostableligase enzyme to circularize the amplification probe.

In accordance with the present invention, further embodiments of in situLD-PCR may utilize amplification probes that are designed to detectvarious genotypic variants of a pathogen e.g. HCV, that are based on theknown HCV sequences of these variants (Stuyver et al., 1993, J. Gen.Vir. 74:1093-1102). For example, different type-specific probes may beadded together to the sample, and detection of HCV sequences andamplification of the probe sequences carried out by in situ LD-PCR asdescribed above. Next, the amplified probe sequences are assayed for thepresence of individual variant genotypes by in situ hybridization withtype specific internal probes that are labeled to facilitate detection.

In certain aspects of the invention, the target nucleic acid sequencemay be directly detected using the various amplification probes and/oramplification sequences described above, without amplification of thesesequences. In such aspects, the amplification probes and/oramplification sequences may be labeled so that they are detectable.

In an embodiment of the invention the RAM amplification method describedherein may be used in a gel matrix format or slide format combined withfluorescent primers to detect aneusomy or gene mutation in situ in asingle cell. Embedding single cells in a gel matrix allows for enzymaticmanipulation of the cell, i.e., proteinase digestion to release DNA,without the lose of genomic material. The gel matrix also protects theDNA from shearing damage and allows for maintenance of the cell'soriginal three dimensional configuration.

The probe hybridization, ligation, and amplification may be carried outin a gel matrix such as polyacrylamide or agarose (See, for example,Dubiley S. et al., 1999, Nucleic Acids Research 27:i-iv). The largemutimeric amplicons generated by primer extension amplification and/orsubsequent ramification amplification can be visualized with afluorescent microscope. Because the gel matrix prevents diffusion, anypositive signal will appear as distinct “dots”. Alternatively, the boundRAM probe can be detected using the hybridization signal amplificationmethod (HSAM).

In embodiments of the present invention utilizing a ligation dependentcircularizable probe, the resulting circular molecule may beconveniently amplified by the ramification-extension amplificationmethod (RAM), as depicted in FIG. 19. Amplification of the circularizedprobe by RAM adds still further advantages to the methods of the presentinvention by allowing up to a million-fold amplification of thecircularized probe under isothermal conditions. RAM is illustrated inFIG. 19.

The single, full length, ligation dependent circularizable probe usefulfor RAM contains regions at its 3′ and 5′ termini that are hybridizableto adjacent but not contiguous regions of the target molecule. Thecircularizable probe is designed to contain a 5′ region that iscomplementary to and hybridizable to a portion of the target nucleicacid, and a 3′ region that is complementary to and hybridizable to aportion of the target nucleic acid adjacent to the portion of the targetthat is complementary to the 5′ region of the probe. The 5′ and 3′regions of the circularizable probe may each be from about 20 to about35 nucleotides in length. In a preferred embodiment, the 5′ and 3′regions of the circularizable probe are about 25 nucleotides in length.The circularizable probe further contains a region designated as thelinker region. In a preferred embodiment the linker region is from about30 to about 60 nucleotides in length. The linker region is composed of ageneric sequence that is neither complementary nor hybridizable to thetarget sequence.

The circularizable probe suitable for amplification by RAM is utilizedin the present method with one or more capture/amplification probes, asdescribed hereinabove. When the circularizable probe hybridizes with thetarget nucleic acid, its 5′ and 3′ termini become juxtaposed. Ligationwith a linking agent results in the formation of a closed circularmolecule.

Amplification of the closed circular molecule is effected by adding afirst extension primer (Ext-primer 1) to the reaction. Ext-primer 1 iscomplementary to and hybridizable to a portion of the linker region ofthe circularizable probe, and is preferably from about 15 to about 30nucleotides in length. Ext-primer 1 is extended by adding sufficientconcentrations of dNTPs and a DNA polymerase to extend the primer aroundthe closed circular molecule. After one revolution of the circle, i.e.,when the DNA polymerase reaches the Ext-primer 1 binding site, thepolymerase displaces the primer and its extended sequence. Thepolymerase thus continuously “rolls over” the closed circular probe toproduce a long single strand DNA, as shown in FIG. 19.

The polymerase useful for amplification of the circularized probe by RAMmay be any polymerase that lacks 3′→5′ exonuclease activity, that hasstrand displacement activity, and that is capable of primer extension ofat least about 1000 bases. (Exo-)Klenow fragment of DNA polymerase,Thermococcus litoralis DNA polymerase (Vent (exo′) DNA polymerase, NewEngland Biolabs) and phi29 polymerase (Blanco et al., 1994, Proc. Natl.Acad. Sci. USA 91:12198) are preferred polymerases. Thermus aquaticus(Taq) DNA polymerase is also useful in accordance with the presentinvention. Contrary to reports in the literature, it has been found inaccordance with the present invention that Taq DNA polymerase has stranddisplacement activity.

Extension of Ext-primer 1 by the polymerase results in a long singlestranded DNA of repeating units having a sequence complementary to thesequence of the circularizable probe. The single stranded DNA may be upto 10 Kb, and for example may contain from about 20 to about 100 units,with each unit equal in length to the length of the circularizableprobe, for example about 100 bases. As an alternative to RAM, detectionmay be performed at this step if the target is abundant or the singlestranded DNA is long. For example, the long single stranded DNA may bedetected at this stage by visualizing the resulting product as a largemolecule on a polyacrylamide gel.

In the next step of amplification by RAM, a second extension primer(Ext-primer 2) is added. Ext-primer 2 is preferably from about 15 toabout 30 nucleotides in length. Ext-primer 2 is identical to a portionof the linker region that does not overlap the portion of the linkerregion to which Ext-primer 1 is complementary. Thus each repeating unitof the long single stranded DNA contains a binding site to whichExt-primer 2 hybridizes. Multiple copies of the Ext-primer 2 thus bindto the long single stranded DNA, as depicted in FIG. 19, and areextended by the DNA polymerase. The primer extension products displacedownstream primers with their corresponding extension products toproduce multiple displaced single stranded DNA molecules, as shown inFIG. 19. The displaced single strands contain binding sites forExt-primer 1 and thus serve as templates for further primer extensionreactions to produce the multiple ramification molecule shown in FIG.19. The reaction comes to an end when all DNA becomes double stranded.

The DNA amplified by RAM is then detected by methods known in the artfor detection of DNA. Because RAM results in exponential amplification,the resulting large quantities of DNA can be conveniently detected, forexample by gel electrophoresis and visualization for example withethidium bromide. Because the RAM extension products differ in sizedepending upon the number of units extended from the closed circularDNA, the RAM products appear as a smear or ladder when electrophoresed.In another embodiment, the circularizable probe is designed to contain aunique restriction site, and the RAM products are digested with thecorresponding restriction endonuclease to provide a large amount of asingle sized fragment of one unit length i.e., the length of thecircularizable probe. The fragment can be easily detected by gelelectrophoresis as a single band. Alternatively, a ligand such as biotinor digoxigenin can be incorporated during primer extension and theligand-labeled single stranded product can be detected as describedhereinabove.

The RAM extension products can be detected by other methods known in theart, including, for example, ELISA, as described hereinabove fordetection of PCR products.

In other embodiments of the present invention, the RAM assay is modifiedto increase amplification. In one embodiment, following the addition ofExt-primer 2, the reaction temperature is periodically raised to about95° C. The rise in temperature results in denaturation of doublestranded DNA, allowing additional binding of Ext-primers 1 and 2 andproduction of additional extension products. Thus, PCR can beeffectively combined with RAM to increase amplification, as depicted inFIG. 16.

In another embodiment, the Ext-2 primer (and thus the identical portionof the linker region of the circularizable probe) is designed to containa promoter sequence for a DNA-dependent RNA polymerase. RNA polymerasesand corresponding promoter sequences are known in the art, and disclosedfor example by Milligan et al. (1987) Nucleic Acid Res. 15:8783.

In a preferred embodiment the RNA polymerase is bacteriophage T3, T7, orSP6 RNA polymerase. Addition of the Ext-primer 2 containing the promotersequence, the corresponding RNA polymerase and rNTPs, allowshybridization of Ext-primer 2 to the growing single-stranded DNA to forma functional promoter, and transcription of the downstream sequence intomultiple copies of RNA. This embodiment of the invention is illustratedin FIG. 17. In this embodiment, both RAM and transcription operate toproduce significant amplification of the probe. The RNA can be detectedby methods known to one of ordinary skill in the art, for example,polyacrylamide gel electrophoresis, radioactive or nonradioactivelabeling and detection methods (Boehringer Mannheim), or the Sharpdetection assay (Digene, Md.). Detection of the RNA indicates thepresence of the target nucleic acid.

In another embodiment, Ext-primer 1 and the corresponding part of thelinker region of the circular probe are designed to have a DNA-dependentRNA polymerase promoter sequence incorporated therein. Thus whenExt-primer 1 binds the circularized probe, a functional promoter isformed and the circularized probe acts as a template for RNAtranscription upon the addition of RNA polymerase and rNTPs. Thedownstream primer and its RNA sequence are displaced by the RNApolymerase, and a large RNA polymer can be made. The RNA polymer may bedetected as described hereinabove. Alternatively, the circular probe canbe cleaved into a single stranded DNA by adding a restriction enzymesuch as EcoRI. The restriction site is incorporated into the 5′ end ofextension primer 1, as shown in FIG. 20.

In the methods described above RAM amplification is used to amplify theprobe. However, modification of the design of the Amp-probe-2 may beused to amplify target sequences. In such instances, the 3′ and 5′ endof the Amp-probe-2 are separated by the target sequences that areintended to be amplified (FIG. 23). The sequences may range in size froma few nucleotides to several thousand nucleotides. The gap between the3′ end and the 5′ end of the probe will be filled using a DNA polymerasewhich lacks 5′-3′ exonuclease and displacement activities. Suchpolymerases are well known to those skilled in the art and include butare not limited to T4 DNA polymerase and modified polymerases lackingexonuclease activity. If the target nucleic acid is RNA, the gap betweenthe 3′ end and the 5′ end of the probe will be filled using reversetranscriptase. Following extension, the gap is closed in with ligase andamplification of the DNA is performed using an ext-primer 1 to generatea long single stranded DNA. Addition of a second primer, ext-primer 2allows amplification of the single stranded DNA by RAM as describedabove.

As described above, the methods of the invention may be used in assaysto specifically detect infectious pathogenic agents and normal andabnormal genes. The present invention further provides methods forgeneral amplification of total genomic DNA or mRNA expressed within acell. The use of such methods provides a means for generating increasedquantities of DNA and/or mRNA from small numbers of cells. Suchamplified DNA and/or mRNA may then be used in techniques developed fordetection of infectious agents, and detection of normal and abnormalgenes.

To amplify genomic DNA, a genomic DNA sample is prepared from cellsusing any of a variety of different methods well known in the art. Onceisolated, the genomic DNA sample is digested with a selected restrictionendonuclease. Restriction endonucleases that may be utilized fordigestion of genomic DNA include, for example, any of those variousenzymes commercially available. After digestion of genomic DNA, adouble-stranded amp-probe is added to the reaction. The amp-probe is adouble stranded DNA fragment of approximately 70-130 nucleotidescontaining a protruding sequence complimentary to the restrictionendonuclease site of the digested genomic DNA. The amp-probe is designedto contain multiple primer sites that will be used to RAM amplify thegenomic DNA. In instances where multiple restriction endonucleases areused to digest the DNA, multiple Amp-probes will be added withprotruding sites complimentary to the different restriction sites. Afterannealing the amp-probes, ligase is added to the reaction to ligate theamp-probe sequences to the fragmented genomic DNA. This process may berepeated a number of times to ensure complete digestion of genomic DNA.

In an embodiment of the invention, to reduce the possibility of adaptorself-ligation, a first strand amp-probe may be added to the reactioncontaining the digested genomic DNA followed by ligation of the firststrand amp-probe to the genomic DNA. Following a wash step to remove theunligated first strand amp-probe, a second strand amp-probe, which willhybridize to the complementary sequences of the first strand amp-probe,is added. Ligase is added to the reaction a second time, resulting ingenomic DNA fragments containing double stranded amp-probes ligated toeach end.

The length of the amp-probe sequence can be increased by repeateddigestion of the DNA fragments with the selected restrictionendonuclease and repeated hybridization, washing and ligation steps.Because the opposite end of the amp-probe is designed to contain arestriction endonuclease site, digestion with the restrictionendonuclease will create a new site for the first amp-probe to bind to.The process can be repeated multiple times thereby increasing theamp-probe length and thus increasing the number of RAM primer bindingsites.

Following addition of the amp-probe, the genomic DNA is denatured andRAM primers designed to bind to sequences within the amp-probe areadded. DNA polymerase and dNPTs are added to the reaction and RAMmediated amplification is initiated. The DNA polymerase to be used inthe amplification reaction is preferably one with a strong displacementactivity and high processivity, such as, for example, Φ29 or Bst DNApolymerase.

In an embodiment of the invention, the addition of amp-probes to theends of the digested genomic DNA can be initially performed in a gelmatrix to ensure the integrity of the DNA fragments and that all theends contain an amp-probe sequence. The efficiency of the amplificationstep is dependent on the number of primer binding sites available in theamp-probe sequence. Thus, for efficient amplification multiple primerbinding sites should be available within the amp-probe sequences. TheDNA fragments can be removed from the gel matrix and subsequentamplification carried out in a reaction vessel. The advantage thismethod of general genomic amplification provides over other PCR basedmethods is the absence of a requirement for multiple cycling and itensures that all DNA fragments are amplified.

Total mRNA may also be amplified using the RAM techniques of the presentinvention. Cellular mRNAs may be purified using methods well known forisolation of RNA including but not limited to capture onto supportmatrices, such as magnetic beads, or nitrocellulose membranes usingoligo (dT) Capture/Amp-probe-1 probes. The Capture/Amp-probe-1 isdesigned to contain an anchor sequence followed by a stretch of 20nucleotides of T which is followed by a RAM primer binding sequence.Reverse transcription by incubation with a reverse transcriptase resultsin generation of a single stranded cDNA. The single stranded cDNA isthen converted to dsDNA using methods well known to those of skill inthe art. A second dsDNA AMP-probe-2 is ligated to the 5′ end of thecDNA. The resulting total cDNA is then amplified as described above forgenomic DNA.

The present invention also provides a novel method for analyzingdifferential mRNA expression patterns between cells, referred to hereinas differential display RAM (DD-RAM). The method involves (i) reversetranscription of mRNA using a 5′ Capture/Amp probe-1 sequence as primer;(ii) ligation of the 3′ end of the extended sequence to the 5′ end of aArbitrary/Amp probe-2 annealed to the mRNA; (iii) RAM amplificationusing a set of RAM primers (forward and reverse primers); and (iv)electrophoretic separation of the resulting fragments. The resultingfragments from different types of cells are compared to identifydifferentially expressed mRNAs. The method of the invention may furthercomprise digestion of the resulting cDNA with a restriction endonucleasethat recognizes a site in the primer.

In addition to the 3′ complementary region, each 5′ Capture/Amp-probewill contain a generic sequence for RAM primers to bind and, forexample, a biotin moiety at the probe 5′ end. The 5′ Capture/Amp probe-1is designed to bind to the 3′ end of the mRNA and will serve both as acapture probe for mRNA isolation and primers for reverse transcription.The 3′ Arbitrary/Amp probe-2 is designed to contain a 5′ degenerativesequence for binding to the 5′ end of the mRNA and a generic sequencefor RAM primers to bind.

In a specific embodiment of the invention, following hybridization ofthe probes with mRNA, the probe/mRNA complex is isolated by capture ontoa support matrix, such as magnetic streptavidin beads via biotin, oroligo (dT) nitrocellulose through the 5′ anchor probes. Extensive washesare performed to remove any unbound probe and cellular DNA. Addition ofreverse transcriptase results in production of a first strand cDNA whichterminates at the 5′ end of the Arbitrary/Amp probe-2. Ligation joinsthe two fragments, i.e., the 5′ end of the Arbitrary/Amp probe-2 and theextended sequence, which then serve as template for subsequent RAMamplification.

To increase the assay sensitivity, a subtraction step may be performedbefore reverse transcription is performed. For subtraction, primers12-15 nucleotides in length and complementary to known housekeepingand/or structural gene sequences are added to the hybridization mix. Theprimers are designed to bind to the 3′ region of the housekeeping and/orstructural mRNAs with a few nucleotides overlapping with the anchorprobe, thereby, competing with the Capture/Amp probe-1 for binding tomRNA. For example, 12-15 nucleotide long primers synthesized tocomplement the 3′ end of housekeeping and/or structural mRNAs such askeratin, laminin, tubulin, acetyl-coenzyme, adenosine deaminase,adenylate kinase, and aldolase A will be added to the hybridization mix.Before adding reverse transcriptase, the reaction is incubated with anRNA specific enzyme which specifically cleaves the RNA strand of anRNA-DNA duplex. Such enzymes, include for example, RNases such asRnaseH. The RNase treatment is designed to eliminate the large number ofhighly expressed housekeeping mRNAs thereby increasing the sensitivityof the assay.

In addition a single probe may be designed to comprise a 5′ anchorsequence and a 5′ arbitrary sequence. The probe may be labeled with abinding moiety, such as biotin, to facilitate isolation of the hybridmolecules from the reaction mixture (for example, using streptavidinbeads). A reverse transcriptase reaction is carried out to extend theregion between both ends of the primer followed by ligation to formclosed circular molecules which can be subsequently amplified by RAM.After digestion with a restriction endonuclease, the resulting productscan be examined on a sequencing gel.

The present invention provides advantages over other types ofdifferential display methods in that (i) each mRNA has only onecorresponding RAM product because only the first available 3′Arbitrary/Amp-probe will be ligated to the extended sequence, therefore,reducing the redundant presentation of the same mRNA; (ii) all ligatedprobes are amplified by the same pair of primers, therefore, minimizingdifferent primer amplification efficiencies; and (iii) with the additionof a subtraction step, housekeeping and/or structural mRNAs areeliminated from the reaction, thus increasing assay sensitivity andspecificity.

The DD-RAM techniques described herein can be utilized to identify mRNAsthat are differentially expressed within different cell types. Forexample, the technique will permit rapid screening of large numbers oftumor cells at different stages of tumorgenesis thereby providing amethod for the identification of important genes that are closelyrelated to tumorogenesis.

Reagents for use in practicing the present invention may be providedindividually or may be packaged in kit form. For example, kits might beprepared comprising one or more first, e.g., capture/amplification-1probes and one or more second, e.g., amplification-probe-2 probes,preferably also comprising packaged combinations of appropriate genericprimers. Kits may also be prepared comprising one or more first, e.g.,capture/amplification-1 probes and one or more second, full length,ligation-independent probes, e.g., amplification-probe-2. Still otherkits may be prepared comprising one or more first, e.g.,capture/amplification-1 probes and one or more second, full length,ligation-dependent circularizable probes, e.g., amplification-probe-2.Such kits may preferably also comprise packaged combinations ofappropriate generic primers. Optionally, other reagents required forligation (e.g., DNA ligase) and, possibly, amplification may beincluded. Additional reagents also may be included for use inquantitative detection of the amplified ligated amplification sequence,e.g., control templates such as an oligodeoxyribonucleotidecorresponding to nanovariant RNA. Further, kits may include reagents forthe in situ detection of target nucleic acid sequences e.g. in tissuesamples. The kits containing circular probes may also includeexonuclease for carryover prevention.

The arrangement of the reagents within containers of the kit will dependon the specific reagents involved. Each reagent can be packaged in anindividual container, but various combinations may also be possible.

The present invention is illustrated with the following examples, whichare not intended to limit the scope of the invention.

Example 1 Detection of HIV-1 RNA in a Sample Preparation ofOligonucleotide Probes

A pair of oligodeoxyribonucleotide probes, designatedCapture/Amp-probe-1 (HIV) and Amp-probe-2 (HIV), respectively fordetecting the gag region of HIV-1 RNA were prepared by automatedsynthesis via an automated DNA synthesizer (Applied Biosystems, Inc.)using known oligonucleotide synthetic techniques. Capture/Amp-probe-1(HIV) is an oligodeoxyribonucleotide comprising 59 nucleotides and a 3′biotin moiety, which is added by using a 3′-biotinylated nucleosidetriphosphate as the last step in the synthesis. The Capture/Amp-probe-1(HIV) used in this Example has the following nucleotide sequence (alsolisted below as SEQ ID NO. 1):

    1          11         21 5′- CCATCTTCCT GCTAATTTTA AGACCTGGTA    31         41         51     ACAGGATTTC CCCGGGAATT CAAGCTTGG -3′

The nucleotides at positions 24-59 comprise the generic 3′ end of theprobe. Within this region are recognition sequences for SmaI (CCCGGG),EcoRI (GAATTC) and HindIII (AAGCTT) at nucleotides 41-46, 46-51 and52-57, respectively. The 5′ portion of the sequence comprisingnucleotides 1-23 is complementary and hybridizes to a portion of the gagregion of HIV-1 RNA.

Amp-probe-2 (HIV) is a 92 nucleotide oligodeoxyribonucleotide which hasthe following sequence (also listed below as SEQ ID NO. 2):

    1          11         21         31         415′- GGGTTGACCC GGCTAGATCC GGGTGTGTCC TCTCTAACTT TCGAGTAGAG    51         61         71         81         91    AGGTGAGAAA ACCCCGTTAT CTGTATGTAC TGTTTTTACT GG -3′

The nucleotides at positions 71-92 comprise the 3′ specific portion ofthis probe, complementary and hybridizable to a portion of the gagregion of HIV-1 RNA immediately adjacent to the portion of the gagregion complementary to nucleotides 1-23 of Capture/Amp-probe-1 (HIV).Nucleotides 1-70 comprise the generic 5′ portion of Amp-probe-2 (HIV).

Ligation of the 5′ end of Capture/Amp-probe-1 (HIV) to the 3′ end ofAmp-probe-2 (HIV) using T₄ DNA ligase creates the ligated amplificationsequence (HIV) having the following sequence (also listed below as SEQID NO. 3):

    1          11         21         315′- GGGTTGACCC GGCTAGATCC GGGTGTGTCC TCTCTAACTT    41         51         61         71    TCGAGTAGAG AGGTGAGAAA ACCCCGTTAT CTGTATGTAC    81         91         101        111    TGTTTTTACT GGCCATCTTC CTGCTAATTT TAAGACCTGG    121        131        141        151    TAACAGGATT TCCCCGGGAA TTCAAGCTTG G -3′

This ligated amplification sequence is 151 nucleotides long, whichprovides an ideal sized template for PCR.

The generic nucleotide sequences of the ligated amplification sequence(HIV) comprising nucleotides 116-135 (derived from nucleotides 24-43 ofCapture/Amp-probe-1 (HIV)) and nucleotides 1-70 (derived fromnucleotides 1-70 of Amp-probe-2 (HIV)) correspond in sequence tonucleotides 1-90 of the (−) strand of the WSI nanovariant RNA describedby Schaffner et al., J. Molec. Biol. 117:877-907 (1977). WSI is one of agroup of three closely related 6 S RNA species, WSI, WSII and WSIII,which arose in Qβ replicase reactions without added template. Schaffneret al. termed the three molecules, “nanovariants.”

The 90 nucleotide long oligodeoxyribonucleotide corresponding tonucleotides 1-90 of the WSI (−) strand has the following sequence (alsolisted below as SEQ ID NO. 4):

    1          11         21         31         415′- GGGTTGACCC GGCTAGATCC GGGTGTGTCC TCTCTAACTT TCGAGTAGAG    51         61         71         81    AGGTGAGAAA ACCCCGTTAT CCTGGTAACA GGATTTCCCC -3′

Two generic oligodeoxynucleotide primers were also synthesized for usein PCR amplification of the ligated amplification sequence. Primer-1,which has a length of 21 nucleotides, is complementary to the 3′sequence of Capture/Amp-probe-1 (HIV) (nucleotides 38-58) and has thesequence (also listed below as SEQ ID NO. 5):

    1          11 5′- CAAGCTTGAA TTCCCGGGGA A -3′

Primer-2, which has a length of 20 nucleotides, corresponds in sequenceto the 5′ sequence of Amp-probe-2 (HIV) (nucleotides 1-20) and has thesequence (also listed below as SEQ ID NO. 6):

    1          11 5′- GGGTTGACCC GGCTAGATCC -3′

Capture and Detection of HIV-1 RNA

Target HIV-1 RNA (100 μl) is dissolved in an equal volume of lysisbuffer comprising 5M GnSCN, 100 mM EDTA, 200 mM Tris-HCl (pH 8.0), 0.5%NP-40 (Sigma Chemical Co., St. Louis, Mo.), and 0.5% BSA in a 1.5 mlmicrofuge tube. Next, the 3′-biotinylated Capture/Amp-probe-1 (HIV) (SEQID NO. 1) and Amp-probe-2 (HIV) (SEQ ID NO. 2), together withstreptavidin-coated paramagnetic beads (obtained from Promega Corp.)were added to the lysed sample in the lysis buffer. A complex comprisingtarget RNA/Capture/Amp-probe-1 (HIV)/Amp-probe-2 (HIV)/paramagneticbeads was formed and retained on the beads. A magnetic field generatedby a magnet in a microfuge tube holder rack (obtained from PromegaCorp.) was applied to the complex to retain it on the side of thereaction tube adjacent the magnet to allow unbound material to besiphoned off. The complex was then washed twice with a 1.5M GnSCN bufferto remove any unbound proteins, nucleic acids, and probes that may betrapped with the complex. The magnetic field technique facilitated thewash steps. The GnSCN then was removed by washing the complex with 300mM KCl buffer (300 mM KCl, 50 mM Tris-HCl, pH 7.5, 0.5% Non-IDEP-40 1 mMEDTA).

The two probes were then covalently joined using T₄ DNA ligase(Boehringer Manheim) into a functional ligated amplification sequence(HIV) (SEQ ID NO. 3), which can serve as a template for PCRamplification. The ligation reaction was carried out in the presence ofa 1× ligation buffer comprising a 1:10 dilution of 10× T₄ DNA ligaseligation buffer (660 mM Tris-HCl, 50 mM MgCl₂, 10 mM dithioeryritol, 10mM ATP-pH 7.5 at 20° C.) obtained from Boehringer Manheim.

The paramagnetic beads containing bound ligated amplification sequence(HIV) were washed with 1× T₄ DNA ligase ligation buffer and resuspendedin 100 μl of 1× T₄ DNA ligase ligation buffer. 20 μl of bead suspensionwas removed for the ligation reaction. 2 μl T₄ DNA ligase was added tothe reaction mixture, which was incubated at 37° C. for 60 minutes.

For PCR amplification of the bound ligated amplification sequence (HIV),80 μl of a PCR reaction mixture comprising Taq DNA polymerase, the twogeneric PCR primers (SEQ ID NOS. 5 and 6), a mixture of deoxynucleosidetriphosphates and ³²P-dCTP was added to the ligation reaction. A twotemperature PCR reaction was carried out for 30 cycles in which hybridformation and primer extension was carved out at 65° C. for 60 secondsand denaturation was carried out at 92° C. for 30 seconds.

After 30 cycles, 10 μl of the reaction mixture was subjected toelectrophoresis in a 10% polyacrylamide gel and detected byautoradiography (FIG. 3, Lane A). As a control, nanovariant DNA (SEQ IDNO. 4) was also subjected to 30 cycles of two temperature PCR, under thesame conditions as for the ligated amplification sequence (HIV),electrophoresed and autoradiographed (FIG. 3, Lane B). As can be seenfrom FIG. 3, the amplified ligated amplification sequence (HIV) migratedin a single band (151 nucleotides) at a slower rate than the amplifiednanovariant DNA (90 nucleotides). The results also indicated thatunligated first and second probes were either not amplified or detected.

Example 2 Direct Detection of HIV-1 RNA in a Sample

The ability of the present method to directly detect the presence ofHIV-1 RNA in a sample was also determined. The probes used in thisExample are the same as in Example 1 (SEQ ID NOS. 1 and 2). For directdetection, Amp-probe-2 (HIV) (SEQ ID NO. 2) was labeled at its 5′ endwith ³²P by the T₄ phosphokinase reaction using ³²P-γ-ATP. The variousreaction mixtures were as follows:

Streptavidin-coated paramagnetic beads, 3′-biotinylatedCapture/Amp-probe-1 (HIV) (SEQ ID NO. 1), Amp-probe-2 (HIV) (SEQ ID NO.2) 5′(³²P), HIV-1 RNA transcript.

Streptavidin-coated paramagnetic beads, 3′-biotinylatedCapture/Amp-probe-1 (HIV), Amp-probe-2 (HIV) 5′(³²P).

Streptavidin-coated paramagnetic beads, Amp-probe-2 (HIV) 5′(³²P), HIV-1RNA transcript.

Hybridizations, using each of the above three reaction mixtures, werecarried out in 200 of a 1M GnSCN buffer comprising 1M GnSCN, 0.5% NP-40(Nonidet P-40, N-lauroylsarcosine, Sigma Chemical Co., St Louis, Mo.),80 mM EDTA, 400 mM Tris HCl (pH 7.5) and 0.5% bovine serum albumin.

The reaction mixtures were incubated at 37° C. for 60 minutes. Afterincubation, the reaction mixtures were subjected to a magnetic field asdescribed in Example 1 and washed (200 μl/wash) two times with 1M GnSCNbuffer and three times with a 300 mM KCl buffer comprising 300 mM KCl,50 mM Tris-HCl (pH 7.5), 0.5% NP-40 and 1 mM EDTA. The amount of³²P-labeled Amp-probe-2 (HIV) that was retained on the paramagneticbeads after washing is reported in Table 1 as counts-per-minute (CPM).The results indicate that, only in the presence of both target HIV RNAand Capture/Amp-probe-1 (HIV), is a significant amount of Amp-probe-2retained on the beads and detected by counting in a β-scintillationcounter.

TABLE 1 Capture of target HIV RNA with Capture/Amp-probe-1(HIV) CPM CPM(after 2 washes (after 3 washes Reaction Mixture with 1M GnSCN) with0.3M KCl) 1. 6254 5821 2. 3351 2121 3. 3123 2021

Example 3 Detection of Mycobacterium Avium-Intracellulaire (MAI) inPatient Samples

A recent paper (Boddinghaus et al., J. Clin. Microbiol. 28:1751, 1990)has reported successful identification of Mycobacteria species anddifferentiation among the species by amplification of 16S ribosomal RNAs(rRNAs). The advantages of using bacterial 16S rRNAs as targets foramplification and identification were provided by Rogall et al., J. Gen.Microbiol., 136:1915, 1990 as follows: 1) rRNA is an essentialconstituent of bacterial ribosomes; 2) comparative analysis of rRNAsequences reveals some stretches of highly conserved sequences and otherstretches having a considerable amount of variability; 3) rRNA ispresent in large copy numbers, i.e. 10³ to 10⁴ molecules per cell, thusfacilitating the development of sensitive detection assays; 4) thenucleotide sequence of 16S rRNA can be rapidly determined without anycloning procedures and the sequence of most Mycobacterial 16S rRNAs areknown.

Three pairs of Capture/Amp-probe-1 and Amp-probe-2 probes are preparedby automated oligonucleotide synthesis (as above), based on the 16S rRNAsequences published by Boddinghaus et al., and Rogall et al. The firstpair of probes (MYC) is generic in that the specific portions of thefirst and second probes are hybridizable to 16S RNA of all Mycobacteriaspp: this is used to detect the presence of any mycobacteria in thespecimen. The second pair of probes (MAV) is specific for the 16S rRNAof M. avium, and the third pair of probes (MIN) is specific for the 16SrRNA of M. intracellulaire. The extremely specific ligation reaction ofthe present method allows the differentiation of these two species at asingle nucleotide level.

A. The probes that may be used for generic detection of all Mycobacterspp. comprise a first and second probe as in Example 1. The first probeis a 3′ biotinylated-Capture/Amp-probe-1 (MYC), anoligodeoxyribonucleotide of 54 nucleotides in length with the followingsequence (also listed below as SEQ ID NO. 7):

    1          11         21         315′- CAGGCTTATC CCGAAGTGCC TGGTAACAGG ATTTCCCCGG     41         51    GAATTCAAGC TTGG -3′

Nucleotides 1-18, at the 5′ end of the probe are complementary to acommon portion of Mycobacterial 16S rRNA, i.e., a 16S rRNA sequencewhich is present in all Mycobacteria spp. The 3′ portion of the probe,comprising nucleotides 19-54 is identical in sequence to the 36nucleotides comprising the generic portion of Capture/Amp-probe-1 (HIV)of Example 1.

The second probe is Amp-probe-2 (MYC), an oligodeoxyribonucleotide of 91nucleotides in length, with the following sequence (also listed below asSEQ ID NO. 8):

    1          11         21         315′- GGGTTGACCC GGCTAGATCC GGGTGTGTCC TCTCTAACTT    41         51         61         71    TCGAGTAGAG AGGTGAGAAA ACCCCGTTAT CCGGTATTAG     81         91    ACCCAGTTTC C -3′

Nucleotides 71-91 at the 3′ end of the probe are complementary to acommon portion of 16S rRNA adjacent the region complementary tonucleotides 1-18 or Capture/Amp-probe-1 (MYC) disclosed above, common toall Mycobacteria spp. Nucleotides 1-70 at the 5′ end of the probecomprise the same generic sequence set forth for Amp-probe-2 (HIV) inExample 1.

End to end ligation of the two probes, as in Example 1, provides ligatedamplification sequence (MYC), 145 nucleotides in length, for detectionof all Mycobacteria spp., having the following sequence (also listedbelow as SEQ ID NO. 9):

    1          11         21         315′- GGGTTGACCC GGCTAGATCC GGGTGTGTCC TCTCTAACTT    41         51         61         71    TCGAGTAGAG AGGTGAGAAA ACCCCGTTAT CCGGTATTAG    81         91         101        111    ACCCAGTTTC CCAGGCTTAT CCCGAAGTGC CTGGTAACAG    121        131        141     GATTTCCCCG GGAATTCAAG CTTGG -3′

B. The pair of probes for specific detection of M. avium are as follows:

The first probe is a 3′ biotinylated-Capture/Amp-probe-1 (MAV), anoligodeoxyribonucleotide of 56 nucleotides in length with the followingsequence (also listed below as SEQ ID NO. 10):

    1          11         21         315′- GAAGACATGC ATCCCGTGGT CCTGGTAACA GGATTTCCCC     41         51    GGGAATTCAA GCTTGG -3′

Nucleotides 1-20 at the 5′-end are complementary to a portion of 16SrRNA specific for M. avium. Nucleotides 21-56 comprise the same genericsequence, as above.

The second probe is Amp-probe-2 (MAV), an oligodeoxyribonucleotide of 90nucleotides in length, with the following sequence (also listed below asSEQ ID NO. 11):

    1          11         21         315′- GGGTTGACCC GGCTAGATCC GGGTGTGTCC TCTCTAACTT    41         51         61         71    TCGAGTAGAG AGGTGAGAAA ACCCCGTTAT CGCTAAAGCG     81     CTTTCCACCA -3

Nucleotides 71-90 at the 3′ end of the probe comprise the specificnucleotide sequence complementary to a region of 16S rRNA specific forM. avium, adjacent the specific sequence recognized byCapture/Amp-probe-1 (MAV). Nucleotides 1-70 comprise the same genericsequence as above.

End to end ligation of the two probes provides a 146 nucleotide longligated amplification sequence (MAV) for detection of M. avium havingthe following sequence (also listed below as SEQ ID NO. 12):

    1          11         21         315′- GGGTTGACCC GGCTAGATCC GGGTGTGTCC TCTCTAACTT    41         51         61         71    TCGAGTAGAG AGGTGAGAAA ACCCCGTTAT CGCTAAAGCG    81         91         101        111    CTTTCCACCA GAAGACATGC ATCCCGTGGT CCTGGTAACA    121        131        141     GGATTTCCCC GGGAATTCAA GCTTGG -3′

C. The pair of probes for specific detection of M. intracellulaire areas follows:

The first probe is a 3′-biotinylated Capture/Amp-probe-1 (MIN), anoligonucleotide of 56 nucleotides in length with the following sequence(also listed below as SEQ ID NO. 13):

    1          11         21         315′- AAAGACATGC ATCCCGTGGT CCTGGTAACA GGATTTCCCC     41         51    GGGAATTCAA GCTTGG -3′

Nucleotides 1-20 at the 5′ end are complementary to a portion of 16SrRNA specific for M. intracellulaire. Nucleotides 21-56 comprise thesame generic sequence as above.

The second probe is Amp-probe-2 (MIN), an oligodeoxyribonucleotide or 90nucleotides in length, with the following sequence (also listed below asSEQ ED NO. 14):

    1          11         21         315′- GGGTTGACCC GGCTAGATCC GGGTGTGTCC TCTCTAACTT    41         51         61         71    TCGAGTAGAG AGGTGAGAAA ACCCCGTTAT CGCTAAAGCG     81     CTTTCCACCT-3′

Nucleotides 71-90 at the 3′ end of the probe comprise the specificnucleotide sequence complementary to a region of M. intracellulaire 16SrRNA adjacent the specific sequence recognized by Capture/Amp-probe-1(MIN).

End to end ligation of the two probes provides a 146 nucleotide longligated amplification sequence (MIN) for detection of M.intracellulaire, having the following sequence (also listed below as SEQID NO. 15):

    1          11         21         315′- GGGTTGACCC GGCTAGATCC GGGTGTGTCC TCTCTAACTT    41         51         61         71    TCGAGTAGAG AGGTGAGAAA ACCCCGTTAT CGCTAAAGCG    81         91         101        111    CTTTCCACCT AAAGACATGC ATCCCGTGGT CCTGGTAACA    121        131        141     GGATTTCCCC GGGAATTCAA GCTTGG -3′

D. In order to detect the presence of the above Mycobacteria spp.,patients' blood specimens are collected in Pediatric Isolator Tubes(Wampole Laboratories, N.J.). The Isolator's lysis centrifugationtechnique enables separation of blood components, followed by lysis ofleukocytes to improve recovery of intracellular organisms (Shanson etal., J. Clin. Pathol. 41:687, 1988). After lysis, about 120 μl ofconcentrated material is dissolved in an equal volume of the 5M GnSCNbuffer of Example 1. The mixture is boiled for 30 minutes, which becauseof the nature of mycobacterial cell walls, is required for lysis ofMycobacteria spp. The subsequent procedures (i.e., capture, ligation,PCR and detection) are the same as those employed in Example 1.

Before the PCR amplification, a direct detection is made by measuringradioactivity representing ³²P-5′-AMP-probe-2 captured on the magneticbeads. After the unbound radioactively-labeled Amp-probe-2 is removed byextensive washing, the target 16S rRNA molecules that are present inconcentrations of more than 10⁶/reaction is detectable. Target 16S rRNAthat cannot be detected directly is subjected to PCR amplification ofthe ligated amplification sequences as per Example 1. The primers foruse in amplification are the same two generic primers of Example 1 (SEQID NOS. 5 and 6).

Example 4 Detection of HCV RNA in a Sample

Hepatitis C virus (HCV), an RNA virus, is a causative agent of posttransfusion hepatitis. HCV has been found to be distantly related toflavivirus and pestivirus and thus its genome has a 5′ and a 3′untranslated region (UTR) and encodes a single large open reading frame(Lee et al., J. Clin. Microbiol. 30:1602-1604, 1992). The present methodis useful for detecting the presence of HCV in a sample.

A pair of oligodeoxynucleotide probes, designated Capture/Amp-probe-1(HCV) and Amp-probe-2 (HCV), respectively, for targeting the 5′ UTR ofHCV RNA are prepared as in Example 1.

Capture/Amp-probe-1 (HCV), which is biotinylated at the 3′ end, is a 55nucleotide long oligodeoxyribonucleotide having the following nucleotidesequence (also listed below as SEQ ID NO. 16):

    1          11         21         315′- GCAGACCACT ATGGCTCTCC CTGGTAACAG GATTTCCCCG     41         51    GGAATTCAAG CTTGG -3′

Nucleotides 1-19 at the 5′ end of Capture/Amp-probe-1 (HCV) comprise aspecific sequence complementary to a portion of the 5′ UTR of the HCVgenome. Nucleotides 20-55 at the 3′ end of the probe comprise the same36 nucleotide generic sequence as in Capture/Amp-probe-1 (HIV) ofExample 1.

Amp-probe-2 (HCV) is a 90 nucleotide long oligodeoxyribonucleotidehaving the following nucleotide sequence (also listed below as SEQ IDNO. 17):

    1          11         21         315′- GGGTTGACCC GGCTAGATCC GGGTGTGTCC TCTCTAACTT    41         51         61         71    TCGAGTAGAG AGGTGAGAAA ACCCCGTTAT CCGGTGTACT     81     CACCGGTTCC-3′

Nucleotides 71-90 comprise the 3′ specific portion of the probe,complementary and hybridizable to a portion of the HCV 5′ UTRimmediately adjacent to the portion of the HCV genome hybridizable tonucleotides 1-19 of Capture/Amp-probe-2 (HCV). Nucleotides 1-70 comprisethe same generic sequence as in Amp-probe-2 (HIV) of Example 1.

End to end ligation of the two probes as in Example 1 provides a 145nucleotide long ligated amplification sequence (HCV) for detection ofHCV in a sample, having the sequence (also listed below as SEQ ID NO.18):

    1          11         21         315′- GGGTTGACCC GGCTAGATCC GGGTGTGTCC TCTCTAACTT    41         51         61         71    TCGAGTAGAG AGGTGAGAAA ACCCCGTTAT CCGGTGTACT    81         91         101        111    CACCGGTTCC GCAGACCACT ATGGCTCTCC CTGGTAACAG    121        131        141     GATTTCCCCG GGAATTCAAG CTTGG -3′

The ligated amplification sequence (HCV) is amplified using a twotemperature PCR reaction as in Example 1. The PCR primers used foramplification are the same two generic primers (SEQ ID NOS. 5 and 6) ofExample 1.

Example 5 Use of Multiple Capture and Amplification Probes to Detect HCVRNA IN a Sample

A pair of amplication probes and two capture/amplification probes wereused to assay for and detect HCV RNA in a sample, thereby increasing thecapture efficiency of the assay.

The capture/amplification probes Capture/Amp-probe-1 (HCV A) (alloligomers described in this Example are designated “(HCV A)” todistinguish from the probes “(HCV)” of Example 4) having SEQ ID NO. 22and Capture/Amp-probe-1A (HCV A) having SEQ ID NO. 23 are designed andsynthesized such that their 5′ termini are biotinylated and the 3′region of the probes comprises sequences complementary to andhybridizable with sequences in the 5′UTR of HCV RNA (FIG. 4). Thegeneric nucleotide sequence at the 5′ region of the probes that are nothybridizable to the target nucleic acid sequence are designed andsynthesized to have random sequences and a GC content of, at least, 60%,and such that they exhibit minimal secondary structure e.g. hairpin orfoldback structures.

Capture/Amp-probe-1 (HCV A) which is biotinylated at the 5′ terminus, isa 45 nucleotide DNA oligomer, such that nucleotides 5 to 45 in the 3′region, are complementary to and hybridizable with sequences in the5′UTR of the target HCV RNA, and that the oligomer has the followingnucleotide sequence (also listed below as SEQ ID NO. 22):

5′- AAGAGCGTGA AGACAGTAGT TCCTCACAGG GGAGTGATTC ATGGT -3′

Capture/Amp-probe-1A (HCV A) which is also biotinylated at the 5′terminus, is also a 45 nucleotide DNA oligomer, such that nucleotides 5to 45 in the 3′ region are complementary to and hybridizable withsequences in the 5′UTR of HCV RNA that are immediately adjacent to theregion of the 5′UTR of the HCV RNA hybridizable with Capture/Amp-probe-1(HCV A) and such that the oligomer has the following nucleotide sequence(also listed below as SEQ ID NO. 23):

5′- AAGACCCAAC ACTACTCGGC TAGCAGTCTT GCGGGGGCAC GCCCA -3′

The two amplification probes Amp-probe-2 (HCV A) and Amp-probe-2A (HCVA) each contain a nucleotide sequence complementary to and hybridizablewith the conserved 5′UTR of HCV RNA.

Amp-probe-2 (HCV A) is a 51 nucleotide oligomer such that nucleotides 1to 30 in the 5′ region are complementary to and hybridizable withsequences in the 5′UTR of HCV RNA, and that the nucleotides 34 to 51 atthe 3′ terminus bind to and hybridize with PCR primer-3 and such thatthe oligomer has the following nucleotide sequence (also listed below asSEQ ID NO. 24):

5′- ACTCACCGGT TCCGCAGACC ACTATGGCTC GTTGTCTGTG TATCTGCTAA C -3′

Amp-probe-2A (HCV A) is a 69 nucleotide oligomer such that nucleotides40 to 69 in the 3′ region are complementary to and hybridizable withsequences in the 5′UTR of HCV RNA genome immediately adjacent to theportion of the HVC RNA genome hybridizable to nucleotides 1-30 ofAmp-probe-2 (HCV A) and such that the nucleotides 1 to 18 at the 5′terminus bind to and hybridize with PCR primer-4 and such thatnucleotides 19 to 36 at the 5′ terminus bind to and hybridize with PCRprimer-5, and such that the oligomer has the following nucleotidesequence (also listed below as SEQ ID NO. 25):

5′- CAAGAGCAAC TACACGAATT CTCGATTAGG TTACTGCAGAGGACCCGGTC GTCCTGGCAA TTCCGGTGT -3′

End to end ligation of the two probes provides a 120 nucleotide ligatedproduct, the ligation-amplification sequence (HCV A) that serves as adetectable sequence for HCV as well as a template for amplificationreactions, and has the sequence (also listed below as SEQ ID NO. 26):

5′- CAAGAGCAAC TACACGAATT CTCGATTAGG TTACTGCAGAGGACCCGGTC GTCCTGGCAA TTCCGGTGTA CTCACCGGTTCCGCAGACCA CTATGGCTCG TTGTCTGTGT ATCTGCTAAC -3′

Primer-3, used for the first series of PCR amplification of the ligatedamplification sequence, SEQ ID NO. 26 (HCV A), and which has a length of18 nucleotides, is complementary to sequence comprising nucleotides 34to 51 at the 3′ terminus of Amp-probe-2 (HCV A), and is, therefore, alsocomplementary to the sequence comprising nucleotides 103 to 120 of theligated amplification sequence, SEQ ID NO. 26 (HCV A), and has thesequence (also listed below as SEQ ID NO. 27):

5′- GTTAGCAGAT ACACAGAC -3′

Primer-4, used for the first series of PCR amplification of the ligatedamplification sequence (HCV A), SEQ ID NO. 26, and which has a length of18 nucleotides, is complementary to the sequence comprising nucleotides1-18 at the 5′ terminus of the Amp-probe-2A (HCV A), and is, therefore,also complementary to the sequence comprising nucleotides 1 to 18 of theligated amplification sequence, SEQ ID NO. 26 (HCV A), and has thesequence (also listed below as SEQ ID NO. 28):

5′- CAAGAGCAAC TACACGAA -3′

Primer-5, a DNA oligomer of 18 nucleotides is used for the second seriesof PCR amplification of the ligated amplification sequence (HCV A), SEQID NO. 26, such that the primer is complementary to the sequencecomprising nucleotides 19-36 of the Amp-probe-2A (HCV A), and is,therefore, also hybridizable to the sequence comprising nucleotides19-36 of the ligated amplification sequence SEQ ID NO. 26 (HCV A), andhas the sequence (also listed below as SEQ ID NO. 29):

5′- TTCTCGATTA GGTTACTG -3′

The assay utilizing the above probes and primers was used to detect HCVRNA in 24 human serum samples that were stored at −70° C. until use. Forthe assay, 180 μl serum sample was added to concentrated lysis buffer(prepared by condensing 250 μl of the lysis solution containing 5MGnSCN, 0.5% bovine serum albumin, 80 mM EDTA, 400 mM Tris HCl (pH 7.5),and 0.5% Nonidet P-40 so that the mixture of serum and lysis bufferwould have a final concentration of 5M GnSCN) mixed well and incubatedfor 1 hour at 37° C. to release the target RNA from HCV particles. 80 μlthe lysis mixture was then transferred to 120 μl of hybridization buffer[0.5% bovine serum albumin, 80 mM EDTA, 400 mM Tris-HCl (pH 7.5), 0.5%Nonidet-P40] with 10¹⁰ molecules each of amplification probes,Amp-probe-2 (HCV A) and Amp-probe-2A (HCV A) oligomers, and 10¹¹molecules each of capture/amplification probes, Capture/Amp-probe-1 (HCVA) and Capture/Amp-probe-1A (HCV A). The addition of the hybridizationbuffer reduced the concentration of the guanidium isothiocyanate (GnSCN)from 5M to 2M to allow the hybridization to occur. The mixture wasincubated at 37° C. for 1 hour to let the various probes hybridize withthe target RNA, whereupon 30 μl of streptavidin coated paramagneticbeads (Promega) were added to the hybridization mixture beforeincubation at 37° C. for 20 minutes to allow ligand binding. Next, thebeads were washed with 150 μl of 2M GnSCN to eliminate any free probes,proteins, extraneous nucleic acids and potential PCR inhibitors from thehybridization mixture; this was followed by the removal of the GnSCN bywashing twice with 150 μl ligase buffer [66 mM Tris-HCl (pH 7.5) 1 mMDTT, 1 mM ATP, 0.5% Nonidet P-40 and 1 mM MnCl₂]. At each wash-step, themagnetic separation of the bound complex from the supernatant waseffected by the magnetic field technique described in Example 1.

The amplification probes, Amp-probe-2 (HCV A) and Amp-probe-2A (HCV A),bound to target RNA were then covalently joined to create the ligatedamplification sequence (HCV A) that was utilized as a template for PCRamplification. The hybrid complex was resuspended in 20 μl ligase bufferwith 5 units of T₄ DNA ligase 1(Boehringer) and incubated for 1 hour at37° C. for the ligation reaction. For the subsequent PCR reactionreferred to hereafter as the “first PCR reaction”, 10 μl of the ligatedmixture, including the beads, was added to 20 μl of PCR mixture [0.06 μMeach of Primer-3 and Primer-4, 1.5 Units Taq DNA Polymerase, 0.2 mM eachof dATP, dCTP, dGTP and dTTP, 1.5 mM MgCl₂, 10 mM Tris-HCl (pH 8.3) 50mM KCl] and the mixture incubated at 95° C. for 30 seconds, 55° C. for30 seconds and 72° C. for 1 minute, for 35 cycles. After the first PCRreaction, 5 μl of the product was transferred to a second PCR mixture[same components as the first PCR mixture except that Primer-4 wassubstituted with Primer-5] for “the second PCR reaction” (a semi-nestedPCR approach to increase the sensitivity of the assay) carried out underthe same conditions as the first PCR reaction. 10 μl of the products ofthe second reaction were electrophoresed on a 6% polyacrylamide gel,stained with ethidium bromide and visualized under ultraviolet light.

To establish the sensitivity and the specificity of the method, 10-foldserial dilutions of synthetic HCV RNA in HCV-negative serum were assayedaccording to the protocol described above, so that the concentration ofHCV RNA ranged from 10 to 10⁷ molecules/reaction. After ligation andamplification, the PCR products were separated by polyacrylamide gelelectrophoresis, stained with ethidium bromide and visualized underultra violet light. The results, shown in FIG. 8, clearly indicate thespecificity of the method. In the absence of HCV RNA there is no signal,indicating that probes must capture the target RNA in order to generatea PCR product. As few as 100 molecules of HCV RNA/sample were detectablewith the semi-nested PCR method (FIG. 8), indicating that thesensitivity of the method is at least comparable to that of conventionalRT-PCR (Clementi et al., 1993, PCR 2: 191-196).

Further, relative amounts of the PCR product represented by theintensity of the bands as visualized in FIG. 8, were proportional to thequantity of the target RNA (HCV RNA transcripts). Therefore, the assayis quantitative over, at least, a range of 10² to 10⁵ target molecules.

To determine the increased capture efficiency afforded by two captureprobes, ³²P-labeled target HCV RNA was assayed for capture and retentionon paramagnetic beads using Capture/Amp-probe-1 (HCV A) orCapture/Amp-probe-1A (HCV A) or both. The capture was estimated by theamount of radioactivity retained on the paramagnetic beads afterextensive washes with 2M-GnSCN buffer and the ligase buffer. Resultsshowed that 25.7% of labeled HCV RNA was retained on the beads whencaptured by Capture/Amp-probe-1 (HCV A) alone, 35.8% retained withCapture/Amp-probe-1A (HCV A) alone and 41.5% of the target RNA wasretained when both the capture probes were used. Therefore thedouble-capture method was more efficient than the use of a singlecapture probe.

Example 6 Use of Multiple Capture and Amplification Probes to DetectHIV-1 RNA in a Sample

An alternative approach to that set forth in Example 1 uses acapture/amplification probe and a pair of amplication probes to detectthe presence of HIV-1 RNA. Capture/Amp-probe-1 (HIV), SEQ ID NO. 1 and apair of amplification probes Amp-probe-2 (HIV A) (all oligomersdescribed in this Example are designated “(HIV A)” to distinguish fromthe probes “(HIV)” of Example 1) (SEQ ID NO. 19) and Amp-probe-2A (HIVA), (SEQ ID NO. 20), are utilized such that the generic nucleotidesequences of the ligated amplification sequence (HIV A) (SEQ ID NO. 21)comprising nucleotides 1-26 derived from nucleotides 1-26 of Amp-probe-2(HIV A) and nucleotides 86-112 derived from nucleotides 40-65 ofAmp-probe-2A (HIV A) are designed and synthesized to have randomsequences and a GC content of, at least, 60%, and such that they exhibitminimal secondary structure e.g., hairpin or foldback structures.

Amplification probe Amp-probe-2 (HIV A) is a 47 nucleotide DNA oligomersuch that nucleotides 27 to 47 in the 3′ region, are complementary toand hybridizable with sequences in the gag region of the target HIV-1RNA, and that the oligomer has the following nucleotide sequence (alsolisted below as SEQ ID NO. 19):

5′- GGTGAAATTG CTGCCATTGT CTGTATGTTG TCTGTGTATC TGCTAAC -3′

Amplification probe Amp-probe-2A (HIV A) is a 65 nucleotide DNA oligomersuch that nucleotides 1 to 39 in the 5′ region, are complementary to andhybridizable with sequences in the gag region of the target HIV-1 RNA,immediately adjacent to the portion of the HIV-1 RNA genome hybridizableto nucleotides 27-47 of the Amp-probe-2 (HIV A) and that the oligomerhas the following nucleotide sequence (also listed below as SEQ ID NO.20):

5′- CAAGAGCAAC TACACGAATT CTCGATTAGG TTACTGCAGCAACAGGCGGC CTTAACTGTA GTACT -3′

End to end ligation of the two amplification probes provides a 112nucleotide ligated amplification sequence (HIV A) such that the sequenceserves as a detectable sequence for HIV-1 RNA as well as a template foramplification reactions, and has the following sequence (also known asSEQ ID NO. 21)

5′- GGTGAAATTG CTGCCATTGT CTGTATGTTG TCTGTGTATCTGCTAACCAA GAGCAACTAC ACGAATTCTC GATTAGGTTACTGCAGCAAC AGGCGGCCTT AACTGTAGTA CT -3′

Further, the capture, detection and optional amplification of thecaptured ligation product in order to assay for HIV RNA is carried outas described in Example 5. The PCR primers used for amplification arethe same primers-3, 4 and 5 (SEQ ID NOS. 27, 28 and 29) of Example 5.

Example 7 Use of Separate Capture/Amplification Probes and a LigationIndependent, Single Amplification Probe to Detect HCV RNA in a Sample

The assay employs a single ligation independent amplification probe andtwo capture/amplification probes to detect HCV RNA in a sample.

The capture/amplification probes Capture/Amp-probe-1 (HCV A) andCapture/Amp-probe-1A (HCV A) used in this method are the same asdescribed in Example 5.

The amplification probe, Amp-probe-2 (HCV B) (all oligomers described inthis Example are designated “(HCV B)” to distinguish from the probes“(HCV)” of Example 4), SEQ ID NO. 30, is a 100 nucleotide DNA moleculesuch that the sequence represented by nucleotides 39 to 79 in thecentral region of the oligomer is complementary to and hybridizable to aregion in the 5′ UTR of the HCV RNA (FIG. 6), and that the sequencesspanning nucleotides 1-38 in the 5′ terminus and by nucleotides 80-100in the 3′ terminus are designed and synthesized such that they haverandom sequences and a GC content of, at least, 60%, and such that theyexhibit minimal secondary structure e.g. hairpin or foldback structures.Amp-probe-2 (HCV B), also referred to as amplification sequence, has thefollowing sequence (also listed below as SEQ ID NO. 30):

5′- CAAGAGCAAC TACACGAATT CTCGATTAGG TTACTGCAGCGTCCTGGCAA TTCCGGTGTA CTCACCGGTT CCGCAGACCG TTGTCTGTGT ATCTGCTAAC -3′

The capture, detection and the optional amplification of the probesequences was carried out as described in Example 5, except thatprimers-3 and -4, only, were utilized in a single PCR amplificationstep, the second PCR step was omitted, and that the ligation step wasomitted.

Example 8 Use of Separate Capture/Amplification Probes and a Single,Amplifiable, Ligation Dependent Probe to Detect HCV RNA in a Sample

The method in this Example employs the two capture/amplification probesCapture/Amp-probe-1 (HCV A) and Capture/Amp-probe-1A (HCV A) describedin Example 5 and a single amplification probe, Amp-probe-2 (HCV C) (alloligomers described in this Example are designated “(HCV C)” todistinguish from the probes “(HCV)” of Example 4) that hybridizes to thetarget nucleic acid and circularizes upon ligation of its free terminias shown in FIG. 7.

Amp-probe-2 (HCV C) is a 108 nucleotide amplification probe, alsoreferred to as an amplification sequence, such that nucleotides 1-26 inthe 5′ terminus of the oligomer are complementary to and hybridizable toa sequence in the 5′UTR of the target HCV RNA (indicated by (a) in FIG.7) and such that nucleotides 83-108 at the 3′ terminus of the oligomerare complementary to and hybridizable to a sequence in the 5′UTR of thetarget HCV RNA (indicated by (b) in FIG. 7). Moreover, when the probehybridizes with the target HCV RNA, the 3′ and 5′ termini of the probeare placed immediately adjacent to each other (FIG. 7), resulting in theformation of a closed circular molecule upon ligation with a linkingagent, such as DNA ligase. The sequence of Amp-probe-2 (HCV C) is givenas follows (also listed as SEQ ID NO. 31):

5′- CCTTTCGCGA CCCAACACTA CTCGGCTGTC TGTGTATCTGCTAACCAAGA GCAACTACAC GAATTCTCGA TTAGGTTACTGCGCACCCTA TCAGGCAGTA CCACAAGG -3′

Primer-3 (SEQ ID NO. 27), used for the first series of PCR amplificationof the ligated and circularized Amp-probe-2 (HCV C), is an 18 nucleotidelong oligomer that is complementary to the sequence comprisingnucleotides 27 to 45 of Amp-probe-2 (HCV C).

Primer-4 (SEQ ID NO. 28), also used for the first series of PCRamplification of the ligated and circularized Amp-probe-2, is a 18nucleotide long oligomer that is complementary to the sequencecomprising nucleotides 46 to 63 of Amp-probe-2 (HCV C).

The hybridization of the two capture/amplification probes and theamplification probe to target HCV RNA, circularization of theamplification probe upon ligation of its termini and amplification ofthe probe sequences was carried out as described in Example 5, exceptthat primers-3 and -4, only, were utilized in a single PCR amplificationstep, the second PCR step was omitted, and that Amp-probe-2 (HCV C) (SEQID NO. 31) was substituted for the pair of amplification probes,Amp-probe-2 (HCV A) (SEQ ID NO. 24) and Amp-probe-2A (HCV A) (SEQ ID NO.25) utilized in Example 5.

To establish the sensitivity and the specificity of the method, 10-foldserial dilutions of synthetic HCV RNA in HCV-negative serum were assayedaccording to the method to provide standard concentrations of HCV RNAranging from 10³ to 10⁷ molecules/sample. After ligation andamplification, the PCR products were separated by polyacrylamide gelelectrophoresis, stained with ethidium bromide and visualized underultra-violet light.

The results, (FIG. 9, (−): control, no sample), indicate the specificityof the method. The assay is highly specific; in the absence of targetHCV RNA there is no visible signal, indicating that probes must capturethe target RNA in order to generate a PCR product. As seen in FIG. 9, asfew as 10⁴ molecules of HCV RNA/sample were clearly detectable.

Further, relative amounts of the PCR product, represented by theintensity of the bands (FIG. 9), were proportional to the quantity ofthe target RNA (HCV RNA transcripts). Therefore, the assay issignificantly quantitative at least over a range of 10⁴ to 10⁷ targetmolecules.

Example 9 Detection of HCV Target Sequences in Tissue Sample UsingLD-PCR Assay

This example provides a comparison of the ligation-dependent PCR (LDPCR)of the present invention with reverse transcriptase PCR (RT-PCR) for thedetection of HCV sequences in formalin fixed, paraffin embedded (FFPE)liver samples. Twenty-one archival liver specimens of hepatocellularcarcinoma (HCCs) from patients who underwent liver resection ororthotopic liver transplantation between January, 1992 to March, 1995 atthe Mount Sinai Medical Center, New York, N.Y. were selected for thisstudy. Thirteen of these patients were anti-HCV positive and eight werenegative as determined by a second generation enzyme-linked immunoassay(EIA II) (Abbott Diagnostic, Chicago, Ill.). An explanted liver tissuefrom an anti-HCV negative patient with cirrhosis secondary to biliaryatresia was used as control. After surgery, the liver specimens werestored at 4° C. and sectioned within twelve hours. The specimens werefixed in 10% buffered formalin for eight to twelve hours and routinelyembedded in paraffin. The FFPE specimens were stored at room temperaturefor a period of three months up to three years. In addition, snap frozenliver tissues from thirteen of the twenty-two patients, stored at −70°C., were used to resolve discordance between LD-PCR and RT-PCR results.

FFPE specimens (approximately 2-4 cm²) were sectioned on a microtomewith a disposable blade to 10 μm in thickness, and each section wasplaced in a 1.5-ml microcentrifuge tube. To avoid cross contamination,the blades were changed and the holder was cleaned with 10% Chloroxsolution between each sample. The sections were deparaffinized byincubating at 60° C. for 10 minutes in the presence of 1 ml of xylene(Sigma). The xylene was removed by two washes with absolute ethanol. Thespecimens were then dried by vacuum centrifugation or by placing on ahot block at 65° C. for 30 min.

For LD-PCR, the deparaffinized tissues were lysed by incubating at 100°C. for 30 min in 250 μl of lysis buffer containing 5 M guanidiniumthiocyanate (GnSCN) (Fluka), 0.5% bovine serum albumin (Sigma), 80 mMEDTA, 400 mM Tris HCl (pH 7.5), and 0.5% sodium-N-lauroylsarcosine(Sigma) followed by incubating at 65° C. for 30 min. The lysed specimenswere stored at −20° C. until use. The HCV serologic status of allspecimens was blinded to laboratory personnel to avoid bias.

For RT-PCR, the deparaffinized tissues were lysed by incubating at 60°C. for 5 hr in 200 μl of lysis buffer containing 10 mM Tris-HCl (pH8.0), 0.1 mM EDTA (ph 8.0), 2% sodium dodecyl sulfate and 500 μg/mlproteinase K. RNA was purified by phenol and chloroform extractionsfollowed by precipitation with an equal volume of isopropanol in thepresence of 0.1 volume of 3 M sodium acetate. The RNA pellet was washedonce in 70% ethanol, dried and resuspended in 30 μl of sterilediethylpyrocarbonate-treated water. RNA was also extracted from sections(10 nm thickness) of frozen liver tissue obtained from the correspondingpatients using the single step RNA extraction method described byChomczynski et al. (1987) Anal. Biochem. 162: 156.

LD-PCR was performed as follows. Briefly, 80 μl of lysis mixture wereadded to 120 μl of hybridization buffer [0.5% bovine serum albumin, 80mM EDTA, 400 Mm Tris-HCl (pH 7.5), and 0.5% sodium-N-lauroylsarcosine],which contained 10¹⁰ molecules of phosphorylated Amp-probe-2, 10¹⁰molecules of Amp-probe 2A and 10¹¹ molecules of capture Amp-probe 1 andcapture Amp-probe 1A. (Probes are as described in Example 5). Additionof the hybridization buffer reduced the GnSCN concentration from 5 M to2 M to allow hybridization to occur. This mixture was incubated for onehour to allow the formation of hybrids, consisting of two DNA captureprobes and two DNA hemiprobes bound to their HCV RNA target. Thirty μlof streptavidin-coated paramagnetic beads (Promega) were added to themixture and incubated at 37° C. for 20 min to allow the hybrids to bindto the bead surface. The beads were then washed twice with 150 μl ofwashing buffer [10 mM Tris-HCl (pH 7.5), 0.5% Nonidet P-40, and 1.5 mMMgCl₂, and 50 mM KCl] to remove nonhybridized probes, as well as GnSCN,proteins, nucleic acids, and any potential PCR inhibitors. During eachwash, the beads were drawn to the wall of the assay tube by placing thetube on a Magnetic Separation Stand (Promega), enabling the supernatantto be removed by aspiration. The hybrids were then resuspended in 20 μlligase solution [66 mM Tris HCl (pH 7.5), 1 mM dithiothreitol, 1 mM ATP,1 mM MnCl₂, 5 mM MgCl₂, and 5 units of T4 DNA ligase (BoehringerMannheim)] and incubated at 37° C. for one hour to covalently link theprobes that are hybridized to adjacent positions on the RNA target, thusproducing the ligated amplification probe described in Example 5. Ten μlof the ligation reaction mixture (including beads) were then transferredto 20 μl of a PCR mixture containing 0.66, μM of PCR primer 3 and 0.66μM of PCR primer 4 as described in Example 5, 1.5 units of Taq DNApolymerase, 0.2 mM dATP, 0.2 mM dCTP, 0.2 mM dGTP, 0.2 mM dTTP, 1.5 mMMgCl₂, 10 mM Tris-HCl (pH 8.3), and 50 mM KCl. The first PCR reactionwas incubated at 90° C. for 30 sec, 55° C. for 30 sec and 72° C. for 1min for 35 cycles in a GeneAmp PCR System 9600 Thermocycler(Perkin-Elmer, Norwalk, Conn.). After the first PCR, 5 μl of eachreaction mixture were transferred into a 30-μl second PCR mixturecontaining the same components except that 0.66 μM of PCR primer 3 and0.66 μM of PCR primer 5 were used for semi-nested PCR. The second PCRreaction was performed by the same protocol as the first PCR reaction.Ten μl of the second PCR reaction were analyzed by electrophoresisthrough a 6% polyacrylamide gel and visualized by ultravioletfluorescence after staining with ethidium bromide. The presence of a 102basepair band for the second PCR product was considered as a positiveresult. All tests were duplicated and done blindly to the serologicalstatus (anti-HCV positive or negative) of the sample.

RT-PCR was performed according to the method of Abe et al. (1994)International Hepatology Communication 2: 352. Briefly, 15 μl of RNAsuspension of each specimen was used as template to detect HCV RNA andbeta actin RNA. The beta actin RNA was used internal positive controlfor cellular RNA. The sequence of outer primers used for RT-PCR are, forHCV RNA, 5′-GCGACACTCCACCATAGAT-3′ (sense) (SEQ ID NO: 32) and5′-GCTCATGGTGCACGGTCTA-3′ (antisense) (SEQ ID NO: 33) and for beta-actinRNA, 5′-CTTCTACAATGAGCTGCGTGTGGCT-3′ (sense) (SEQ ID NO: 34) and5′-CGCTCATTGCCAATGGTGATGACCT-3′ (antisense) (SEQ ID NO: 35). Thesequence of inner primers are, for HCV RNA, 5′-CTGTGAGGAACTACTGTCT-3′(sense) (SEQ ID NO: 36) and 5′-ACTCGCAAGCACCCTATCA-3′ (antisense) (SEQID NO: 37), and for beta-actin RNA, 5′-AAGGCCAACCGCGAGAAGAT-3′ (sense)(SEQ ID NO: 38) and 5′-TCACGCACGATTTCCCGC-3′ (antisense) (SEQ ID NO:39). The first PCR reaction was combined with the reverse transcriptionstep in the same tube containing 50 μl of reaction buffer prepared asfollows: 20 units of Rnase inhibitor (Promega), 100 units of Moloneymurine leukemia virus reverse transcriptase (Gibco BRL), 100 ng of eachouter primer, 200 μM of each of the four deoxynucleotides, 1 unit of TaqDNA polymerae (Boehringer Mannheim) and 1× Taq buffer containing 1.5 mMMgCl₂. The thermocycler was programmed to first incubate the samples for50 min at 37° C. for the initial reverse transcription step and then tocarry out 35 cycles consisting of 94° C. for 1 mM, 55° C. for 1 min, and72° C. for 2 min. For the second PCR, 5 μl of the first PCR product wasadded to a tube containing the second set of each inner primer,deoxynucleotides, Taq DNA polymerase and Taq buffer as in the first PCRreaction, but without reverse transcriptase and Rnase inhibitor. Thesecond PCR reaction was performed with the same protocol as the firstPCR reaction but without the initial 50 min incubation at 37° C. Twentyμl of the PCR products were examined by electrophoresis through a 2%agarose gel. Positive results of HCV RNA and beta-actin RNA wereindicated by the presence of second PCR products as a 268-basepair and a307-basepair band, respectively.

The results of LD-PCR and RT-PCR are set forth below in Table 2.

TABLE 2 Comparison of LD-PCR with RT-PCR FFPE^(a) Unfixed^(b) HCVLD-PCR^(c) RT-PCR^(d) RT-PCR^(e) Serology (No) + − + − + − Anti-HCV + 130 5 8 7^(f) 0 (13) Anti-HCV − 5 4 0 9 6^(g) 1 (9) ^(a)FFPE--formalinfixed paraffin embedded liver tissues. ^(b)Unfixed--snap frozen livertissues of corresponding FFPE specimens. ^(c)Number of FFPE specimenstested positive (+) or negative (−) by ligation-dependent PCR.^(d)Number of FFPE specimens tested positive (+) or negative (−) byreverse transcription PCR. ^(e)Number of specimens confirmed by RT-PCRusing unfixed frozen tissues. ^(f)Only 7 unfixed specimens wereavailable for confirmatory 2RT-PCR test. ^(g)Only 7 unfixed specimenswere available for confirmatory RT-PCR test.

Of the twenty-two FFPE specimens, thirteen were obtained from patientswho were HCV positive by EIA assay and nine were HCV negative (Table 2).HCV RNA was detected in all thirteen seropositive FFPE specimens byLD-PCR, whereas only five were positive by RT-PCR. For confirmation,unfixed frozen liver specimens available from seven cases were tested byRT-PCR. Of these seven cases, HCV-RNA was detectable in all seven byLD-PCR when FFPE tissue of the same specimens were utilized, but in onlyone by RT-PCR. However, RT-PCR on the frozen tissue confirmed thepresence of HCV-RNA in all cases. Beta actin mRNA was detected in allcorresponding specimens, indicating minimal RNA degradation. Theseresults confirmed the preservation of the HCV RNA duringformalin-fixation, the heated paraffin embedding process, and up tothree years of storage. The overall sensitivity of RT-PCR on FFPEspecimens was 23.8% (5/21) in this study while it was determined 58.6%and 84% in prior studies by El-Batonony et al. (1994) J. Med. Virol.43:380 and Abe et al. The gross difference in these values was due tothe difference in the selection of specimens in these studies (eightRT-PCR negatives and five positives on FFPE tissues were selected forthis study). Among the eight HCV sero-negative liver specimens, sevenwith HCC were removed from two patients with primary biliary cirrhosis(PBC), two with alcoholic cirrhosis, two with hepatitis B virus (HBV)liver cirrhosis, one with cryptogenic liver cirrhosis and one withoutHCC from a child with biliary atresia (Table 3). Among the seven HCCliver specimens, five tested positive for HCV by LD-PCR, but none byRT-PCR. The specimen with biliary atresia remained negative by both PCRtests. To resolve this discrepancy, RT-PCR was performed on the sevenunfixed frozen tissue specimens. The results are set forth below inTable 3.

TABLE 3 HCV RNA detected in HCV-seronegative cases Total ClinicalFFPE^(b) Unfixed^(c) confirmed Diagnosis (No)^(a) LD-PCR^(d) RT-PCR^(d)RT-PCR^(e) Positive PBC (2) 1 0 2 2 Alcoholic (2) 2 0 2 2 Biliaryatresia (1) 0 0 N/D 0 HBV (3) 2 0  2^(g) 2 Cryptogenic (1) 0 0 0 0^(a)Liver specimens from patients with various clinical diagnosis:PBC—primary biliary cirrhosis, Alcoholic—alcoholic liver cirrhosis,HBV—positive for HBsAg, Cryptogenic—Cryptogenic liver cirrhosis.^(b)FFPE—formalin fixed paraffin embedded liver tissues.^(c)Unfixed—snap frozen, unfixed liver tissues of corresponding FFPEspecimens. ^(d)Number of FFPE specimens tested positive for HCV RNA byLD-PCR or RT-PCR. ^(e)Number of specimens confirmed by RT-PCR usingunfixed frozen tissues. ^(g)Only 2 unfixed specimens were available forconfirmatory RT-PCR test. N/D: not done—no fresh frozen specimenavailable.

The RT-PCR results on unfixed tissue confirmed the LD-PCR results,indicating false negative results by serologic testing. In addition, oneof the PBC specimens that tested negative by both LD-PCR and RT-PCR onFFPE specimens was positive by RT-PCR on an unfixed frozen specimen,indicating false negative results by both PCRs on the FFPE specimen.These results show that there is a high detection rate of HCV RNA in HCVseronegative HCC (6/8, 75%) (Table 3) and that the overall positive ratein both HCV seropositive and seronegative HCC specimens is 86% (18/21)(Table 2). Contamination was unlikely since the cutting of FFPE andunfixed specimens, and the PCR assays were performed in two separatelaboratories. In addition, great precaution was taken in the specimenpreparation and PCR testing with proper negative controls. The overallagreement between LD-PCR of FFPE specimens and RT-PCR on fresh frozenspecimens is very high, and the sensitivity of LD-PCR is 95% (18/19).

The foregoing results suggest that crosslinks caused by formalinfixation disrupt chain elongation of the nascent DNA strand by reversetranscriptase, resulting in lower sensitivity of RT-PCR in FFPE tissue.In contrast, LD-PCR amplifies probe sequences, bypassing the step ofprimer extension along the cross-linked template. In addition, theamplification probes may only have a 30-nucleotide long complementaryregion, and therefore are more accessible to the non-crosslinkedregions. LD-PCR can thus achieve a higher sensitivity in the detectionof HCV RNA in FFPE specimens. The value of this sensitive assay isconfirmed by the foregoing results, which evidence a high detection rateof HCV RNA even in seronegative specimens.

Example 10 Primer Extension-Displacement on Circular AmplificationSequence

This example demonstrates the ability of Klenow fragment of DNApolymerase to displace downstream strands and produce a polymer.

A synthetic DNA target was detected by mixing 10¹² molecules ofphosphorylated circularizable probe having SEQ ID NO:31 with 10¹³molecules of synthetic HCV DNA target in 10 μ.d of 1× ligation buffer,heating at 65° C. for two minutes, and cooling to room temperature forten minutes. One μl of ligase was added to the mix and incubated at 37°C. for one hour, followed by addition of 10¹³ molecules of ³²P-labeledExt-primer having SEQ ID NO:27. The mixture was heated to 100° C. forfive minutes and then cooled to room temperature for twenty minutes.Forty μl of Klenow mix and dNTPs were added to the reaction andincubated at 37° C. Ten μl aliquots were removed at 0, 1, 2 and 3 hoursand examined on an 8% polyacrylamide gel. The results are shown in FIG.18. The left lanes depict results in the absence of ligase. The rightlanes depict extension after ligation. Bands ranging from 105 to 600bases can be visualized in the right lanes. The results demonstrate thatKlenow is able to extend from the Ext-primer, displace the downstreamstrand, and generate polymers.

Example 11 Detection of EBV Early RNA (Eber-1) in Parotid PleomorphicAdenomas by Ligation Dependent PCR

LD-PCR utilizing a circularized probe was performed to detect EpsteinBarr virus early RNA (EBER-1) in salivary benign mixed tumors (BMT). Sixspecimens of BMT and adjacent parotid tissue, and three specimens ofnormal parotid tissue (two removed from cysts and one from ahyperplastic lymph node) were snap frozen in embedding medium for frozentissue specimens (OCT, Miles, Inc., Elkhart, In.) and liquid nitrogen,and stored at −70° C. The corresponding formalin fixed paraffin embedded(FFPE) blocks of tissue were obtained and studied in parallel to thefresh tissue. All tissue was sectioned on a microtome, the blade ofwhich was cleaned with 10% Chlorox between cases to avoid crosscontamination. Two to three sections of each specimen were placed in a1.5 ml microcentrifuge tube. FFPE tissues were deparaffinized byincubating at 60° C. for 10 minutes with 1 ml xylene (Sigma), which wassubsequently removed by two washes with absolute ethanol. Thesespecimens were dried by placing on a hot block at 65° C. for 30 minutes.Deparaffinized tissue was lysed by incubation at 100° C. for 30 minutes,then 65° C. for 30 minutes in 250 μl of lysis buffer: 5M guanidiumthiocyanate (GTC) (Fluka), 0.5% bovine serum albumin (Sigma), 80 mMEDTA, 400 mM Tris HCl (pH 7.5), and 0.5% sodium-N-lauroylsarcosine(Sigma). Fresh frozen tissue was lysed by incubation at 37° C. for 60minutes in the same lysis buffer. The lysed specimens were stored at−20° C. until use.

Two capture/amplification probes designed to flank the region of EBER-1were used to capture target RNA. The sequences for capture probe 1 (SEDID NO: 40) and capture/amplification probe 2 (SEQ ID NO: 41) are shownin Table 4. The circular amplification probe (SEQ ID NO: 42) wasdesigned with 3′ and 5′ regions complementary to the chosen targetsequence (Table 4). Interposed between these two regions is anoncomplementary linker sequence. This circular amplification probecircularized upon target hybridization in such a manner as to juxtaposethe 5′ and 3′ ends. Seminested PCR was performed using primer pairsdirected at this linker sequence, also shown in Table 4.

TABLE 4 Sequences of Capture Probes, Amplifiable Circular Target Probe,and PCR Primers EBER-Cap/Amp-15′Biotin-AAGAgtctcctccctagcaaaacctctagggcagcgtaggtcctg-3′(SEQ ID No. 40) EBER-Cap/Amp-25′Biotin AAGAggatcaaaacatgcggaccaccagctggtacttgaccgaag-3′(SEQ ID No. 41) Circular Amp PROBE5′tcaccacccgggacttgtacccgggacTGTCTGTGTATCTGCTAACCAAGAGCAACTACACGAATTCTCGATTAGGTTACTGCgggaagacaaccacagacaccgttcc-3′(SEQ ID No. 42) 1st PCR GTTAGCAGATACACAGAC primer pairs:(sense SEQ ID NO. 27) CAAGAGCAACTACACGAA (antisense SEQ ID NO. 28)2ND PCR GTTAGCAGATACACAGAC primer pairs: (sense SEQ ID NO. 27)TTCTCGATTAGGTTACTG (antisense SEQ ID NO. 29) (lower case - complementaryto EBER-1, upper case - generically designed)

LD-PCR was performed as follows. Briefly, 80 μl of lysis mixture wereadded to 120 μl of hybridization buffer (0.5% bovine serum albumin, 80mM EDTA, 400 MM Tris-HCl (pH 7.5), and 0.5% sodium-N-lauroylsarcosine(Sigma) which contained 10¹⁰ molecules of phosphorylated target probe,and 10¹¹ molecules of capture probe 1 and capture probe 2. Addition ofthe hybridization buffer reduced the GnSCN concentration from 5 M to 2 Mto allow hybridization to occur. This mixture was incubated for one hourto allow the formation of hybrids, consisting of two DNAcapture/amplification probes and one DNA circular amplification probehybridized on the target RNA. Thirty μl of streptavidin-coatedparamagnetic beads (Promega) were added to the mixture and incubated at37° C. for 20 minutes to allow the hybrids to bond to the bead surface.The beads were washed twice with 150 μl of washing buffer (10 mM TrisHCl (pH 7.5), 0.5% Nonidet P-40, and 1.5 mM MgCl₂ and 50 mM KCl) toremove nonhybridized probes as well as potential inhibitors of PCR (GTC,proteins) and potential sources of nonspecific PCR products (cellularnucleic acids). During each wash, the beads were drawn to the wall ofthe assay tube by placing the tube on a Magnetic Separation Stand(Promega), enabling the supernatant to be removed by aspiration. The 3′and 5′ ends of the circular amplification probes hybridized directlyadjacent to each other on the target RNA, were covalently linked, andhence circularized by incubation at 37° C. for 1 hour with 20 μl ligasesolution (66 mM Tris HCl (pH 7.5), 1 mM dithiothreitol, 1 mM ATP, 1 mMMnCl₂ and 5 units of T4 DNA ligase (Boerhinger)). Ten μl of the ligationreaction mixture, including paramagnetic beads, were transferred to 20μl of a PCR mixture containing 0.66 μM of PCR primer, 0.5 units Tag DNApolymerase, 0.2 mM dATP, 0.2 mM dCTP, 0.2 mM dGTP, 0.2 mM dTTP, 1.5 mMMg_(t), and 10 mM Tris-HCl (pH 8.3) and 50 mM KCl. The first PCRreaction was incubated at 94° C. for 30 seconds, 55° C. for 30 seconds,and 72° C. for 1 minute for 35 cycles in a GeneAmp PCR system 9600thermocycler (Perkin Elmer, Conn.). After the first PCR, 5 μl of eachreaction mixture were transferred into a 25 μl second PCR mixturecontaining the same components except that 0.66 μM of PCR primer 1 and0.66 μM of PCR primer 3 were used for seminested PCR, which increasessignal detection sensitivity without compromising amplificationspecificity. Extension of PCR primer along the covalently circularizedprobe results in the generation of a large multi-unit polymer (rollingcircle polymerization). In fact, without digestion into monomeric units,the PCR polymer product cannot migrate into the polyacrylamide gel. Tenμl of the second PCR reaction were digested with restrictionendonuclease EcoRI in the presence of 50 mM NaCl, 100 mM Tris-HCl (pH7.5), 10 mM MgCl₂, 0.025% Triton X-100, and analyzed by gelelectrophoresis through a 6% polyacrylamide gel and visualized byultraviolet fluorescence after staining with ethidium bromide. Thepresence of a 90 base-pair band (second PCR product) and a 108 base-pairproduct (1st PCR) are considered as a positive result. The results aresummarized in Table 5.

TABLE 5 EBV early RNA (EBER-1) detected by LD-PCR Parotid tissuePleomorphic Adenoma Case (frozen) (frozen) FFPE 1 positive None positive2 negative None negative 3 negative None ND 4 ND positive negative 5positive positive negative 6 positive positive positive 7 positivenegative negative 8 positive positive negative 9 positive negativenegative Note Case 1 and 2 were from parotid tissues removed for reasonsother than pleomorphic adenoma. Cases 3-8 contained pleomorphic adenoma.FFPE—formalin fixed paraffin embedded tissue. Frozen-tissue snap frozenin liquid nitrogen. ND—not done as tissue not available.

In sum, EBER-I sequences were detected in six of eight parotid samples.Of the six pleomorphic adenomas studied, four were positive for EBER-1.Of the two cases in which EBER was not detected in the tumor, sequenceswere present within surrounding parotid tissue. The detection of EBER-1sequences within corresponding formalin-fixed paraffin embedded tissuewas considerably less sensitive—only two of eight specimens werepositive.

In summary, the present results with ligation dependent PCR utilizing acircular probe demonstrate the presence of EBV-related sequences withinthe majority of pleomorphic adenomas studied. The present methodexhibits a markedly increased detection rate relative to standard PCRfor the detection of EBV DNA as performed by Taira et al. (1992) (J. ofOtorhinolaryngol Soc. Jap.) 95: 860. In the present method, the 3′ and5′ ends of a circularizable probe hybridized to the target sequence,resulting in juxtaposition. The juxtaposed sequences were then ligated,resulting in a circularized covalently linked probe that was locked ontothe target sequence and thus resistant to stringent washes. PCR on thecircular probe produced a rolling circle polymer, which was digestedinto monomeric units and visualized on a gel. The use of ligationdependent PCR with a circular probe, followed by detection byamplification of the probe by the rolling circle model, resulted intremendous sensitivity of target detection in fresh frozen tissue.

Example 12 Differential Display Ram

5′ Capture/Amp-probes and 3′ Arbitrary/Amp-probes are designed asfollows. 12 possible 5′ Capture/Amp-probe oligo (dT) probes, used incombination with 24 different 10-mer 3′ Arbitrary/Amp-probes, aresufficient enough to display 10,000 of the mRNA species that are presentin a mammalian cell (Lung et al., 1992, Science 257:967-971). Since theterminal 3′ base of the 5′ capture oligo (dT) probe provides most of theselectivity, the number of capture oligo (dT) probes may be reduced from12 to 3 (Lung et al., Science 1992, 257:967-971; Liang et al., 1994,Nucl. Acid Res. 22:5763-5764).

Initially, three separate 5′ Capture/Amp-probes are synthesized, eachcontaining a nucleotide G, A, or C at the 3′ termini. Adjacent to theterminal nucleotide is a oligo (dT)₁₁ which will serve as both a captureand anchoring sequence. The 5′ region of the Capture/AMP-probes comprisemultiple, i.e., 5-20, generic primer binding sequences with a biotinmoiety at the 5′ end. These multiple primer binding sites are designedfor RAM amplification to ensure sensitivity. If initial tests with threeCapture/Anchor probes do not achieve a good differential display, 4-12separate Capture/Anchor probes can be synthesized based on thecombination of the last two nucleotides (T12MN, M=degenerative A, G, orC; N=A, C, G, and T).

3′ Arbitrary/Amp-probes, 10 nucleotides in length hybridize to mRNA, andproduce enough display bands to be analyzed by a sequencing gel.However, not every probe 10 nucleotides in length is suitable. Probesshould, therefore, be tested experimentally (Bauer, 1993, Nucl. AcidRes. 21:4272-4280). The actual number of 3′ Arbitrary/Amp-probesrequired to display most mRNA species is 24 to 26 different probes.Therefore, initially, 24 3′ Arbitrary/Amp-probes are synthesizedseparately. Each 3′ Arbitrary/Amp-probe contains a 5′ arbitrarysequence, for example 10 nucleotides in length, and a 3′ RAM primerbinding sequence which may be 70-130 nucleotides in length. The 5′ endof each 3′ Arbitrary/Amp-probe is phosphorylated by incubating with T4DNA kinase in order for ligation to occur. The 3′ Arbitrary/Amp-probesare mixed in an equal molar ratio to a final concentration of 10¹¹molecules/μl. The concentration of each 3′ Arbitrary/Amp-probe may bechanged to achieve best differential display.

The DD-RAM assay is carried out as previously described with minormodification (Zhang et al., 1998 Gene 211:277-285; Park, 1996, Amer. J.Path. 149:1485-1491). Tissue sections (5-10 um thickness) or cellsuspensions (1×10⁶ cell/ml) are lysed by incubation at 37° C. for 60minutes in 250 μl of lysis buffer containing 5M guanidium thiocyanate(GTC) (Fluka), 0.5% bovine serum albumin (Sigma Chemical Co., St. Louis,Mo.), 80 mM EDTA, 400 mM Tris HCl (pH 7.5), and 0.5%sodium-N-lauroylsarcosine (Sigma). 80 μl of lysis mixture is added to120 μl of hybridization buffer [0.5% bovine senun albumin, 80 nM EDTA,400 mM Tris-HCl (pH 7.5), and 0.5% sodium-N-lauroylsarcosine], whichcontains 10¹² molecules of each capture/anchored probe and a mixture of10¹¹ molecules of phosphorylated arbitrary sequence probes. Addition ofhybridization buffer reduces the GTC concentration from 5 M to 2 Mthereby allowing hybridization to occur. The hybridization mixture isincubated at 37° C. for one hour to allow the formation of hybrids,consisting of 5′ Capture/Amp-probes and 3′ Arbitrary/Amp-probes bound totheir mRNA targets. 30 μl of streptavidin-coated paramagnetic beads (1mg/ml, Promega, Madison, Wis.) are added to the mixture and incubated at37° C. for 20 min to allow the hybrids to bind to the bead surface. Thebeads are then washed twice with 180 μl of washing buffer [10 mMTris-HCl (pH 7.5), 50 mM KCl, and 1.5 mM MgCl2, and 0.5% Nonidet P-40(Sigma)] to remove nonhybridized probes, as well as GTC, proteins,nucleic acids, and any potential ligation and RAM inhibitors.

The hybrids are then resuspended in 20 μl RT/ligase solution [66 mM TrisHCl (pH 7.5), 1 mM dithiothreitol, 1 nM ATP, 0.2 mM dTAP, 0.2 mM dCTP,0.2 mM dGTP, 0.2 mM dTTP, 1 mM MnCl2, 5 mM MgCl2, and 200 units ofMoloney murine leukemia virus reverse transcriptase (BoehringerMannheim), and 5 units of T4 DNA ligase (Boehringer Mannheim)] (Hsuih,1996) and incubated at 37° C. for one hour to extend from the 5′Capture/Amp-probe to the 3′ downstream arbitrary sequence probes. Thegap between the arbitrary probe and extended sequence is ligated to formcovalently-linked circular probes that can be amplified by a RAM assayas described above. Ten μl of the RT/ligation reaction mixture(including beads) is then transferred to 40 μl of a RAM mixturecontaining 0.66 uM of RAM forward primers and 0.66 uM of RAM reverseprimers, 90 ng of Φ29 DNA polymerase (Boehringer Mannheim), 80 μM³²PdATP, 80 μM dCTP, 80 μM dGTP, 80 μM dTTP, 5 mM MgCl2, and 66 mMTris-HCl (pH 7.5). The RAM reaction is incubated at 35° C. for twohours. If the sensitivity is not enough to display the rare mRNA, 5 μlof the first RAM reaction mixture is transferred into a 25-ul second RAMmixture containing the same components for the second RAM reaction.Fifteen p. 1 of the RAM reaction is analyzed by electrophoresis througha 6% polyacrylamide gel and visualized by autoradiograph.

Example 13 Ram Assay with Multiple Primers

To test whether the addition of multiple RAM primers was able toincrease the efficiency of the RAM reaction, a reaction was performedwith an EBER Amp-probe-2 and three RAM primers. 10¹¹ molecules ofsynthetic EBER DNA target was hybridized with 10¹¹ molecules of EBERAmp-probe-2. Following ligation, one RAM forward primer and two reverseRAM primers (one forward and one reverse), or three RAM primers (oneforward and two reverse) were added to each reaction together with Φ29DNA polymerase.

The products of the reactions were examined on an 8% polyacrylamide gel.Results indicated that with one primer, multimeric ssDNA was producedand that a subset of the products were so large that they did not enterthe gel. Although the amount of product increased with the increasingnumbers of primers used (see, FIG. 21) two primers, lane B; threeprimers, lane C), exponential amplification was not observed. In theabsence of target, no product was observed (lane D), indicating that thereaction is specific.

To increase the efficiency of the reaction, the number of primers wasincreased from 3 to 6 and the length of the primers was shortened from18 nucleotides to 12 nucleotides. Shortening the primer length increasesthe accessibility of the primer to template, while increasing the primernumber drives the equilibrium of the reaction towards hybridization.

Conditions may be further optimized by addition of 6 mM [NH₄]₂SO₄, 10%DMSO and O.5 μg Gene 32 protein to RAM reaction. Under such conditions,10⁴ molecules of EBER targets can be detected (FIG. 23).

As judged by the amount of DNA produced (10′³ molecules of DNA producedfrom 10⁴ molecules of initial Amp-probe-2), a billion-fold amplificationwas achieved. It is noteworthy that reducing primer length did notincrease non-specific background.

Two additional Amp-probe-2 probes were designed to test the efficiencyof the reaction in the presence of six primers. One Amp-probe-2 wassynthesized to contain 3 forward-primer binding sites and 3 reverseprimer binding sites with each primer spaced out by an opposite primer.The second Amp-probe-2 was designed to contain 6 primer binding sites,however, only 2 primer sequences (one forward and one reverse) wereincluded. This particular primer design has the advantage of bothincreasing the hybridization rate while minimizing the interferencebetween primers bound to Amp-probe-2.

Various publications are cited herein, the contents of which are herebyincorporated by reference in their entireties.

1. A method for amplifying a mRNA comprising: (a) hybridizing aCapture/Amp-probe to the 3′ poly-A sequence of a mRNA, wherein theCapture/Amp-probe contains multiple RAM primer sites, wherein the primersite is 10 to 60 nucleotides in length; (b) reverse transcribing of themRNA to generate a single stranded cDNA such that the cDNA contains apoly-T sequence; (c) converting the single stranded cDNA of step (b) todouble stranded DNA, wherein the single strand of DNA of the doublestranded DNA of step (c) which is complementary to the single strandedcDNA of step (b) contains a poly-A sequence; (d) ligating with aligating agent at least one double stranded Amp-probe to one end of thedouble stranded DNA, whereby the Amp-probe is ligated to the endopposite the poly-A sequence in the double stranded DNA, wherein theAmp-probe contains multiple RAM primer sites; (e) denaturing the doublestranded DNA; and (f) amplifying the DNA under isothermal conditions bycontacting the DNA with: (1) at least two RAM primers that arecomplementary and hybridizable to the RAM primer sites; (2) dNTPs; and(3) a DNA polymerase having strand displacement activity, underisothermal conditions whereby the RAM primers are extended from the RAMprimer sites to the end of the DNA to form multiple single strandedDNAs.
 2. The method of claim 1, wherein the DNA polymerase is φ29 DNApolymerase or Bst DNA polymerase.
 3. The method of claim 1, whereinligation of the Amp-probe is performed in a matrix.
 4. The method ofclaim 1, wherein the ligating agent is an enzyme or a chemical agent. 5.The method of claim 4, wherein the enzyme is a DNA ligase.
 6. The methodof claim 5, wherein the DNA ligase is T₄ DNA ligase or Taq DNA ligase.7-16. (canceled)